Vascular Stress Signaling in Hypertension

Abstract

Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. While cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence and pro-inflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy and mitochondrial fission/fusion, signaling effector stimulator of interferon genes (STING)-mediated responses and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.

Introduction

Cells respond to stress by modulating many signaling pathways ranging from cell survival to cell death pathways. Initial responses to a stressful stimulus normally aid in cell recovery from insult to maintain organismal homeostasis^1–3. However, if the stress stimulus persists and/or the cell has a poor adaptive capacity to resolve the insult, the stress responses become stronger, chronic and lead to a secondary (and usually one-way) wave of stress-signaling pathways, including death pathways (apoptosis, necroptosis, pyroptosis) or senescence pathways².

The scenario is more complex if we consider that i) multiple stress-response pathways can be simultaneously activated or recruited at different time points, ii) there is an interplay among signaling pathways, and iii) cell signaling is influenced across tissues, organs or the whole organism by physiological and pathological processes, such as aging and development of cardiovascular diseases (CVD) including hypertension, which is the topic of this compendium.

Accordingly, it has become a consensus that the development of hypertension is a result of chronic physiological maladaptation against various stressors. These include neurohormonal factors [norepinephrine/epinephrine, angiotensin II (Ang II), aldosterone, anti-diuretic hormone or vasopressin], circulating or local bioactive substances derived from immune cells, adipose tissue, skeletal muscle and endothelial cells, mechanical stresses (arterial pressure and shear stress), intracellular accumulation of metabolites (carbon dioxide, hydrogen and potassium ions) and toxic molecules as well as dysfunctional organelles^4–7. In the development of hypertension, chronic maladaptation against these insults eventually leads to disruption of cellular homeostasis, likely contributing to worsened blood pressure control and hypertensive complications. Sedentary lifestyle, excess caloric intake, obesity, hyperglycemia and aging likely exaggerate these processes. It is therefore very important to better understand the molecular mechanisms by which affected organs, such as the vasculature, cope with “hypertensive” stressors at cellular and organelle levels. Taking this opportunity, we will discuss recent advancements in stress-adaptive signaling mechanisms as potential therapeutic targets for people with hypertension and pre-hypertensive conditions such as those with early vascular aging.

Animal Models and Limitations

Animal models of primary hypertension have been developed and extensively reviewed elsewhere⁸. The main experimental models include Ang II infusion, deoxycorticosterone acetate (DOCA)-salt, spontaneously hypertensive rats (SHR), and nitric oxide (NO) synthase inhibition by L-N-nitro arginine methyl ester (L-NAME). The most cited model, chronic Ang II infusion mimics the activation of the renin-angiotensin aldosterone system (RAAS) and generates most of the end-organ injuries seen in human hypertension⁹. Although studies with Ang II infusion have significantly contributed to our understanding of hypertension, the inability to mimic aging and other genetic or environmental stressors seen in human hypertension is a limitation. In rodent models, “adult age” is usually between 6 and 20 weeks, likely contributing to the lack of translation into clinical outcomes⁹. To overcome these limitations, more rigorous studies have begun to combine multiple models such as aging, salt-sensitive hypertension, and metabolic dysfunction in rodents^{10, 11}. In the clinical practice, RAAS blockade, regardless of mono- or combination therapy, does not show the desired level of end-organ protection or reduction in mortality, although it effectively reduces blood pressure^{12, 13}. This indicates that several non-RAAS factors contribute to hypertensive end-organ damage. Obviously, the use of Ang II model alone may lead to incomplete conclusions on the impact of a given stress signaling in hypertension. While most studies cited in this review use a single (Ang II) model, with a few studies using two distinct models, it is important to reconcile the stress mechanisms discussed here with multiple hypertension models for better knowledge as well as for potential translation.

Premature Vascular Aging Associated with Common Stress Signaling

In a disease-free middle-aged population, there is a subset of people whose vasculature demonstrates a significant sign of premature aging, i.e. endothelial dysfunction and arterial stiffness. They are thus considered a high-risk population for future development of hypertension and coronary artery disease. This condition, also known as early vascular aging (EVA) syndrome, is frequently associated with hypertension^{14, 15}. In contrast, there is another chronologically middle-aged population whose vasculature is as healthy as much younger populations [Healthy or Super Normal Vascular Aging (HVA/SUPERNOVA)]¹⁵. This group of people is hypothesized to be protected from CVD as well as other aging-accelerated disorders, such as neurodegenerative diseases, including Alzheimer’s¹⁶. This perspective represents a growing field of study in which ‘biological age’, rather than chronological age, can be therapeutically targeted. Although the molecular mechanisms involved in the development of EVA in humans remain unclear, environmental exposure to several chronic stressors (over nutrition, sedentary lifestyle, smoking, alcohol e.g.) seem to be involved¹⁵. Accordingly, this review article addresses the intracellular stress-sensing and -responding signaling mechanisms that are likely relevant for hypertension development and progression and with potential for add-on therapies.

Both aging (regardless of chronological or biological/EVA) and arterial hypertension are linked to functional, structural and mechanical vascular changes. Characteristic vascular abnormalities in aging and hypertension include endothelial dysfunction, calcification and increased stiffness, remodeling and chronic low-grade inflammation¹⁷. Common mechanisms involved in vascular dysfunction in hypertension and other aging-related CVD likely include (but are not limited to) those that mediate or prolong chronic cell stress, such as 1. endoplasmic reticulum (ER) stress and accumulation of protein aggregates, 2. oxidative stress, 3. metabolic stress centered at mitochondria, 4. DNA damage, 5. stress-induced senescence and senescence-associated secretory phenotype (SASP), and 6. generation of pro-inflammatory factors and processes by danger signals. There also are common adaptive mechanisms against these stresses including unfolded protein responses (UPR), antioxidant response element (ARE) signaling, autophagy, mitophagy and mitochondrial fusion, induction of signaling effector stimulator of interferon genes (STING) pathway and senolysis (elimination of senescent cells).

ER Stress Responses in Hypertension

Hypertensive animal models confirm the presence of aortic ER stress markers and pharmacological inhibition via chemical chaperone 4-phenylbutyric acid (4-PBA) and/or tauroursodeoxycholic acid (TUDCA) reduces fibrosis, inflammation, vascular hypercontractility, and increased blood pressure^18–21. Single cell transcriptomics of hearts from angiotensin II (Ang II)-infused mice identified ER stress elements to be highly involved in hypertensive cardiac remodeling²². Other studies found that Ang II induces protein aggregate formation in the heart²³ and kidney²⁴. At the cellular level, a variety of extracellular stressors and humoral factors, including Ang II, elicit induction of ER stress/UPR markers in endothelial cells (ECs), vascular smooth muscle cells (VSMCs), cardiac myocytes and cardiac fibroblasts²⁵. Moreover, induction of protein aggregate accumulation and UPR were seen in VSMCs stimulated with Ang II²⁶, suggesting that Ang II-induced proteotoxicity in the vasculature is a novel target for hypertension therapy. Enhanced ER stress/UPR within biopsied endothelial cells has been found in obese adults with endothelial dysfunction²⁷. An ongoing clinical trial is testing TUDCA effects on vascular dysfunction in older and obese individuals (Clinicaltrials.gov identifier: NCT04001647). However, how ER dynamics and protein aggregates contribute to hypertension and vascular damage require further investigation.

UPR and ER-Associated Degradation

The ER is an essential organelle for protein synthesis and calcium (Ca²⁺) handling. Chaperones such as HSP47 and glucose-regulated protein 78 (GRP78) aid in properly folding proteins into tertiary structures²⁸. When imbalances in protein synthesis, reduction-oxidation (redox) status, or Ca²⁺ handling occur, three transmembrane ER sensors initiate the stress-induced UPR to maintain homeostasis (proteostasis). These include activating transcription factor 6 (ATF6), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and inositol-requiring transmembrane kinase 1 alpha (IRE1α). Transcription factors induced by UPR activation bind ER stress response elements, leading to the expression of chaperones, ER-associated degradation (ERAD) genes, and autophagy machinery, thus restoring ER protein folding competency²⁹.

Under chronic ER stress however, UPR signaling seems maladapted and likely contributes to hypertension pathology. C/EBP homologous protein (CHOP) activation of caspases and BAX/BIM release of cytochrome C leads to apoptosis³⁰. Ang II infusion increases CHOP and ATF6 in vivo, and global CHOP deficient mice are protected from hypertension-mediated cardiac hypertrophy, fibrosis and vascular dysfunction³¹. In VSMC from SHR, compared with those from WKY, NAPDH oxidases are increasingly localized in the ER, which overall contributes to increased oxidative stress. Moreover, treatment with 4-PBA reduces vascular dysfunction in stroke-prone SHR (SHRSP)¹⁸. In a combination model of hypertensive chronic kidney disease (CKD) utilizing Ang II infusion plus DOCA-salt, 4-PBA treatment not only reduced blood pressure and renal ER stress markers, but significantly attenuated gene expression of inflammatory and fibrotic markers³². These observations suggest that while the UPR is considered a defense mechanism against proteotoxicity, over-activation of UPR is detrimental and contributes to hypertension.

Pharmacological targeting of the individual ER UPR arms, instead of general ER stress, via 4-PBA and TUDCA have yielded promising results. The ATPase vasolin-containing protein (VCP) is involved in ERAD signaling and is reduced in SHR. Overexpression of VCP attenuates Ang II-induced hypertrophy in vitro via mTOR signaling, and reduces cardiac dysfunction associated with pressure overload in mice³³. Overall, VCP activity enhancement to reduce hypertrophy seems to protect against hypertensive remodeling. However, its role in vascular cells remains undetermined. In high-salt diet hypertensive mouse models, vascular expression of the ER stress-dependent transcription factor ATF4 is increased and regulates blood pressure and endothelial function. Furthermore, gut-microbiota composition in salt-sensitive hypertensive mice can be targeted via potassium supplementation to reduce ATF4 expression and, subsequently, blood pressure³⁴. Pharmacological inhibition of IRE1α-XBP1 branch has anti-fibrotic and anti-inflammatory effects via an IRE1α inhibitor, KIRA6 or STF-08310 ^35–37. Overall, small molecule inhibitors targeting each arm of UPR, including the IRE1α UPRsome branch as well as ERAD machinery, are promising opportunities to reverse hypertension and its vascular consequences.

To identify upstream therapeutic targets to reduce hypertensive ER stress, protein aggregation has been explored. Adenoviral GRP78 delivery attenuated Ang II-induced protein aggregate formation and inflammatory monocyte adhesion to VSMCs²⁸. Moreover, pre-amyloid oligomers were formed in VSMCs in response to Ang II stimulation, which were attenuated with 4-PBA treatment²⁶. In hypertension-associated atrial fibrillation, lipid oxidation develops and contributes to amyloid deposition in the heart. Scavenging these reactive peroxidation lipid products via chaperone 3-HOBA reduces atrial amyloid burden and blood pressure in Ang II-infused mice³⁸. These observations suggest a potential application of protein chaperoning therapy against hypertension and related CVD in consonance with clinical trials for age-associated neurodegenerative diseases.

Effects of ER stress inhibition in hypertension have been summarized to allow an overall understanding of therapeutic targets in the vasculature (Figure 1, Table 1). The UPR pathway can be either adaptive or chronic and potentially maladaptive. The adaptive arm has developed to maintain ER function and proteostasis, whereas the chronic arm sustains inflammation, causes organ damage, induces senescence, or eliminates dysfunctional cells via apoptosis. Such maladaptive ER UPR occurs in hypertension due to increased protein synthesis demand via hypertrophic stimuli^{39, 40} and oxidative stress¹⁸. Therefore, restoration of normal proteostasis can protect the vasculature and organs from damage in addition to RAAS inhibition.

Figure 1. Pharmacological targets of ER stress signaling in hypertension.

AT1R signaling disrupts ER homeostasis via increased protein synthesis demand and oxidative stress. Adaptive stress responses including the UPR, ER mediated autophagy (ER-phagy), and ER associated degradation are induced. These responses are initiated by chaperone GRP78 detachment from three transmembrane molecules in order to aid in refolding of misfolded proteins. IRE1α oligomerizes and autophosphorylates to induce RNAse activity to alternatively splice XBP1 mRNA to XBP1s. XBP1s serves as a transcription factor binding to ER stress response elements (ERSE) to induce transcription of cytokines, lipid biogenesis, chaperones, and ERAD. IRE1α also acts as a scaffold for TRAF2 docking and initiation of proinflammatory JNK and NF-κB signaling. PERK dimerizes and autophosphorylates leading to phosphorylation of eIF2α and translation attenuation in an attempt to reduce overwhelming demand of protein folding on the ER. There is an increase translation in ATF4 expression which acts as a transcription factor for amino acid metabolism, autophagy elements, and an antioxidant response. However, CHOP expression is also induced which leads to apoptosis. GRP78 detachment from ATF6 results in its translocation to the Golgi where it is cleaved by S1 and S2 proteases resulting in ATF6 cleaved for which transcribes chaperones, ERAD elements, and XBP1. ERAD induction is mediated by VCP/p97, a Ca²⁺-associated ATPase which participates in ubiquitin-proteasome system to degraded misfolded proteins by interacting with E3 ubiquitin ligases such as HRD1. ER-phagy involves PI3K complexes which form at ER tubules to recruit autophagic initiation complexes, leading to the lipidation of LC3 and formation of autophagosome. Created with BioRender.com

Table 1.

Potential targets in stress-elicited signaling pathways

Potential Targets	Therapeutic approach	Outcome	Reference
Endoplasmic Reticulum	inhibition via 4-PBA or TUDCA)	reduces fibrosis, inflammation, vascular hypercontractility, BP	18–21
		attenuates Ang II-induced formation of pre-amyloid oligomers	26
		prevents Ang II-induced senescence in VSMCs and ECs	26, 92
Unfolded Protein Responses	GRP78 overexpression	protects against I/R injury	230
	adenoviral GRP78 delivery	attenuates Ang II-induced protein aggregate formation and monocyte adhesion to VSMCs	28
	compound 147 (cleaves ATF6)	protects against cardiomyocyte oxidative stress	231
	IRE1α- XBP1 inhibition	anti-fibrotic and anti-inflammatory effects	35–37
	CHOP knockdown	attenuates cardiac hypertrophy, fibrosis and altered vascular reactivity	31
Mitochondrial Stress	SIRT3 overexpression	improves endothelial function and attenuates Ang II-induced hypertension	49
	SIRT3/SOD2 activation	increases endothelial reparative capacity of progenitor cells	50
	chaperone 3-HOBA	decreases vascular dysfunction and hypertension	51
	FAM3A knockdown	reduces Ang II-mediated cardiac and vascular remodeling, vasoconstriction, and hypertension	52
Mitochondrial Fission/Fusion and Mitophagy	17-DMAG (inhibits HSP90)	inhibits Ang II-induced mitochondrial fission and adventitial remodeling	59
	DRP1 (inhibition or knockdown)	protects against Ang II-induced AAA	60
cGAS-STING	STING (inhibition or knockdown)	reduces ATAAD incidence in mice prevents Ang II-induced abnormal vascular reactivity reduces pro-apoptotic, pro-necroptotic, pro-pyroptosis, and pro-inflammatory signaling prevents EC inflammation	71
NRF2	tBHQ (activator of Nrf2)	prevents Ang II-induced endothelial dysfunction, remodeling, and hypertension	116
	Nrf2 deletion	increases sympathetic outflow, impairs baroreflex function and induces hypertension	121
	bardoxolone/L-sulforaphane (NRF2 activators)	improve vascular function and reduces Ang II-induced ROS generation	117
TLR	TLR2 knockdown	prevents impaired vascular tone, iNOS-derived NO formation and TNF-α production prevents Ang II-induced aortic fibrosis and endothelial to mesenchymal transition	158,159
	VIPER (TLR4 blocker)	prevents hypertension induced by abnormal HDL containing symmetric dimethylarginase reduces BP, HMGB1, and TNF-α, IL-1β expression	168,170
	TAK242 (TLR4 inhibitor)	reduces BP and expression of TLR4, MyD88, NF-κB, TNF-α and IL-1β	169
	anti-TLR4	decreases BP and reduces expression of TLR4, COX-2, TxA2, IL-6	171
	TLR4 silencing	attenuates Ang II-induced cardiac hypertrophy	174
	TLR3 silencing	prevents Ang II-induced increased BP and cardiac hypertrophy	174
NLPR3	Canakinumab (human monoclonal IL-1β antibody)	reduces cardiovascular events and hospitalization decreases hypertension and renal fibrosis	211–213, 232
	anakinra (IL-1R antagonist)	prevents hypertension-associated cardiovascular dysfunction and oxidative stress. attenuates hypertension, vascular remodeling,	214
	NLRP3 (gene silencing or blockade)	reduces aldosterone-induced vascular damage	193
	MCC950 (NLRP3 inhibitor)	reduces BP and renal damage and dysfunction in salt-sensitive hypertension	209
		restores EC function and reduces stress oxidative and inflammatory processes	192
		reduces cardiac damage and improves insulin and leptin sensitivity	224, 225, 228

4-PBA, 4-Phenylbutyric acid; AAA, aortic abdominal aneurism; Ang II, angiotensin II; BP, blood pressure; ECs, endothelial cells; FAM3A (family with sequence similarity 3 member A);heat shock protein 90 (HSP90); I/R, ischemia/reperfusion; iNOS, inducible nitric oxide synthase; tBHQ, tert-butylhydroquinone; TUDCA, tauroursodeoxycholic acid; VSMCs, vascular smooth muscle cells.

Autophagy and ER Stress in Hypertension

While autophagic responses are involved in a wide variety of homeostatic and adaptative adjustments against various stresses conditions, autophagy also plays a key role in alteration of ER stress by removing toxic protein aggregates termed aggrephagy. In several CVD models, including Ang II-induced hypertension and cardiovascular remodeling, both pharmacological and genetic manipulation of autophagy have been conducted.

For example, Ang II was infused in heterozygous mice for the autophagy protein 5 (Atg5), an E3 ubiquitin ligase that mediates autophagosome formation. In control wild type mice, Ang II infusion enhanced autophagy, assessed by induction of Atg5, and increased LC3-II/LC3-I ratio. Mitophagy in macrophages was also enhanced in Ang II-infused mice. As expected for the homeostatic roles of autophagy and mitophagy, Ang II-induced cardiac fibrosis was exaggerated in Atg5^+/− mice and this was associated with enhanced reactive oxygen species (ROS) production and NF-κB activation in macrophages⁴¹. In line with these observations, in hearts of a swine model of renovascular hypertension, Ang II type 1 receptor (AT1R) antagonism attenuated markers of enhanced autophagy and mitophagy⁴². However, it remains unclear whether Ang II signaling or hypertension drives autophagy in mice, while Ang II-induced autophagy is observed in VSMCs⁴³. In SHR, supplementation with the autophagy inducer trehalose did not change blood pressure. However, mesenteric resistance arteries displayed preserved endothelial function and reduced arterial stiffness⁴⁴. Under chronic ER stress, ATF4-induced autophagy is known to function at overwhelming ERAD capacity⁴⁵ and this is a part of the common integrated stress response pathways⁴⁶. It is therefore necessary to further evaluate the relationship between UPR and autophagy in hypertension.

Mitochondrial Stress Response in Hypertension

Mitochondria are the powerhouse of cellular and energy metabolism. The mitochondrial outer membrane directly interacts with the ER membrane and regulates fission/fusion events, whereas the inner mitochondrial membrane is particularly involved in oxidative phosphorylation, ATP generation, Ca²⁺ signaling, and protein import. These dynamic functions play critical roles in cell metabolism, antioxidant capacity, inflammation, growth, proliferation, and apoptosis. Accordingly, mitochondrial dysfunction has been identified in both human and animal models of hypertension⁴⁷, however investigations to identify pharmacological targets of intervention are still ongoing.

Mitochondrial Oxidative Stress and the Defense System

Metabolic disorders and oxidative stress contribute to the pathogenesis of vascular dysfunction, including hypertension⁴⁸. During normal cellular respiration, utilization of the mitochondrial electron transport chain (ETC) to synthesize large quantities of ATP³⁶ requires a series of univalent reductions, leading sequentially to the production of superoxide anion (O₂.−).

Antioxidant enzymes have evolved to limit oxidized byproducts from interrupting normal cellular physiology. One such class of enzymes, sirtuins (SIRT) are critical in the redox conversion of O₂.− to hydrogen peroxide (H₂O₂). Arterioles from hypertensive humans have significantly reduced mitochondrial SIRT3, and global SIRT3 depletion enhances vascular O₂.−, blood pressure, hypertrophy, permeability, senescence, and inflammation in Ang II and DOCA-salt mouse models of hypertension⁴⁹. Thus, transgenic SIRT3 overexpression improves endothelial function and attenuates Ang II-induced hypertension, suggesting that this mitochondrial target has therapeutic potential.

Superoxide dismutases (SODs) are part of a major antioxidant defense system, and manganese SOD2 in the mitochondria is regulated by deacetylation via SIRT3 activity. Endothelial progenitor cells from hypertensive patients have impaired reparative capacity, which can be mitigated by SIRT3/SOD2 signaling⁵⁰. Reactive dicarbonyl lipid peroxidation products, isolevuglandins are increased in hypertensive patients, and mitochondria-targeted scavenging of these oxidative stress byproducts using chaperone 3-HOBA has therapeutic potential to treat vascular dysfunction and hypertension⁵¹. A novel mitochondrial protein, FAM3A (family with sequence similarity 3 member A), enhances ATP production and is increased in arteries of hypertensive mice. VSMCs specific FAM3A deletion reduces Ang II-mediated cardiac and vascular remodeling, vasoconstriction, and increased blood pressure in mice⁵². While these observations, summarized at Table 1, support therapeutic potential in manipulating mitochondrial oxidative stress or energy production in hypertension, clinical trials appear to have mixed outcomes.

Interestingly, both Ang II AT1R and AT2R are localized within the mitochondria⁵³, and intracellular Ang II fusion protein selectively expressed in the mitochondria of renal proximal tubules cells regulates ETC complexes, inhibits proximal tubules sodium reabsorption, natriuretic response and contributes to hypertension⁵⁴. These findings have not been explored in other cell types and may represent a new area of hypertension research.

Mitochondrial Fission/Fusion and Mitophagy

Although ATP generation is the primary function of mitochondria, mitochondrial dynamics affect the non-bioenergetic capacity of cells. Fission and fusion of mitochondria control energy homeostasis within the cell while imbalances may contribute to CVD such as obesity, heart failure, and hypertension⁵⁵. The homeostatic balance can lean towards fission to create smaller mitochondria, whereas fusion results in a more interconnected mitochondrial network. Both processes are regulated by guanosine triphosphatases (GTPases) and non-GTPase receptor proteins. For fission, dynamin-related protein 1 (DRP1) is targeted to the outer mitochondrial membrane by mitochondrial fission factor⁵⁶. Excessive fission is a signal of increased mitochondrial damage and leads to enhanced mitophagy, i.e. clearance of damaged mitochondria via autophagosome engulfment and lysosomal degradation⁵⁷. Fusion is mediated by mitofusins 1 and 2 (MFN1 and MFN2) in the outer membrane, and optic atrophy 1 (OPA1) in the inner membrane⁵⁸.

Hypertensive stimuli augment the vascular cell expression of mitochondrial dynamic proteins. In adventitial fibroblasts, Ang II treatment increases mitochondria fission, collagen and α-SMA which can be inhibited with DRP1 silencing⁵⁹. Moreover, pharmacological inhibition of heat shock protein 90 (HSP90) with 17-DMAG blocks Ang II-induced calcineurin regulation of DRP-1-mediated mitochondrial fission and adventitial remodeling⁵⁹. In human abdominal aortic aneurysm (AAA) samples, DRP1 is increased in the aortic medial layer, and pharmacological inhibition of DRP1 by mdivi1, or heterozygous knockdown protects against Ang II-induced AAA in mice⁶⁰. While phosphorylation sites primarily act to induce or inhibit DRP1 oligomerization around mitochondria, a high fat diet has been shown to increase DRP1 acetylation in the heart and to contribute to cardiomyocyte dysfunction and death⁶¹. In ECs, DRP1 activation enhances inflammation and monocyte recruitment⁶², and in macrophages, it promotes intimal thickening after vascular injury⁶³. Moreover, in SHR,4- week mdivi1 treatment reduced hypertension and medial aortic hypertrophy, which were associated with reduction of inflammatory cytokines⁶⁴. Whereas evidence supports that mitochondria fission mediates vascular hypertensive responses, limited studies have explored fusion protein regulation in this process.

Mitophagy is inherently tied to autophagy activation, which has proven beneficial in many CVD treatments⁵³. Evidence supports that Ang II increases mitophagy mediator Parkin in VSMCs, which can aid in the removal of damaged mitochondria⁶⁵. Also in VSMCs, oxidized LDL increases PINK1, ubiquitinated MFN2, and LC3 association to the mitochondria to protect against apoptosis ⁶⁶. In an experimental model of kidney fibrosis via unilateral ureteral obstruction, MFN2 and Parkin were decreased, while LC3-II was also decreased. Furthermore, both Pink1^−/− and Prkn^−/− mice display higher collagen deposition in obstructed kidneys compared to WT mice, via enhanced macrophage TGF-β1 expression⁶⁷. These studies show that deficiencies in the removal of damaged mitochondria can have detrimental effects on cellular death and inflammation. Therefore, suppression of mitochondria damage or enhancing removal pathways via pharmacological tools may yield protection against hypertension.

Mitochondrial DNA Activation of cGAS-STING Signaling

Cytosolic DNA sensing by the cyclic GMP-AMP synthase (cGAS)-STING pathway is implicated in a variety of biological processes associated with intracellular DNA stress. DNA from various sources including the release of mitochondrial DNA (mtDNA) following stress can enter the cytosol and engage the cGAS-STING pathway. Upon activation, STING translocates from the ER to the Golgi, where it recruits kinases TANK- binding kinase 1 (TBK1) and IκB kinase, which phosphorylate interferon regulatory factor 3 (IRF3). Phosphorylated IRF3 dimerizes and translocates to the nucleus to activate transcription of genes encoding type I interferons such as interferon-β (IFNβ)⁶⁸.

The prime function of this pathway is in host defense via the inflammasome activation. However, recent studies have established a link between CVD stressors and the STING pathway. IRF3 is abundantly found in human samples of coronary plaques⁶⁹. Phenylephrine-induced aortic contraction is enhanced in Ang II-infused wild-type mice, but not in STING deficient mice, indicating the involvement of STING signaling in aortic contractile dysfunction⁶⁹. IRF3 reduction is not protective against Ang II infusion-mediated hypertension or cardiac hypertrophy, but it does protect against hypertension-associated cardiac fibrosis⁷⁰. Saturated fatty acid palmitate induces EC inflammation, an effect that appears to be mediated by mtDNA release and IRF3 activation⁷¹. Moreover, mitochondrial damage inducer CCCP and purified mitochondrial DNA increase ICAM1 expression in ECs. Silencing cGAS, STING or IRF3 inhibited palmitic acid-induced mtDNA release and reduced adhesion molecule expression⁷¹. According to these findings, the STING/IRF3 signal induced by damaged mitochondria and subsequent mtDNA release may represent drug targets against vascular inflammation and cardiac fibrosis associated with hypertension.

ER-Mitochondria Communication

Mitochondria are linked to the ER at specialized regions of membrane adherence called mitochondria-associated membranes (MAMs), which facilitate Ca²⁺flux into the ER. Physical linkage between the two organelles occurs via inositol 1,4,5-trisphosphate receptors (IP3R), voltage-dependent anion-selective channel protein (VDAC1) and a chaperone GRP75. Transforming growth factor-β (TGF-β), a key cytokine in hypertension, causes uncoupling of mitochondria from the ER Ca²⁺ release by suppressing IP3R-mediated Ca²⁺ efflux⁷². Furthermore, spatial colocalization of IP₃R to TRPC3 channels enhances vasoconstriction in hypertension⁷³. The MAMs and their dynamic regulation seem understudied in hypertension and warrant further investigation. Figure 2 summarizes mitochondrial stress signaling potentially involved in hypertension and associated CVD.

Figure 2. Pharmacological targets of mitochondrial stress signaling in hypertension.

AT1R signaling induces oxidative stress in the mitochondria. In the vasculature, hypertensive stimuli lead to decreased antioxidant mitochondrial components SIRT3 and SOD2, increased ATP production via enhanced FAM3A and ETC complexes. Increased mitochondrial ROS and damage induces adaptative stress responses. Mitochondrial-associated membranes (MAMs) link mitochondria to the ER. Cellular stress causes a surge of Ca²⁺ via VDAC1 and IP3R which contributes to apoptosis initiation. Mitochondrial damage also leads to the release of mtDNA which can be sensed by cyclic GMP-AMP synthase (cGAS) and its downstream signaling effector stimulator of interferon genes (STING). This leads to NF-κB and IRF3 transcription of interferons and cytokines to contribute to inflammation. Mitophagy via PINK1 depolarization in the inner mitochondrial space leads to Parkin ubiquitin ligase activity on the outer mitochondria membrane which recruits autophagy machinery for phagosome engulfment of damaged mitochondria. Mitochondrial damage also heightens DRP1 GTPase activity to induce fission and tilt the balance away from fusion (mediated via MFN2). Excessive mitophagy and mitochondrial fission has been found in hypertensive models and contributes to vascular pathology. Created with BioRender.com.

Hypertension and Vascular Senescence

As we focus on targeting organelle dysfunction to circumvent hypertensive vascular dysfunction, it is necessary to address other cellular phenotypic changes that organelle homeostasis can mediate. For instance, cell senescence (“to grow old in Latin”) has been characterized in many animal models of CVD^74–77, including those with hypertension^{49, 78–80}. In humans, hypertension positively correlates with age, and hypertensive insults may accelerate the cellular aging process including senescence. Therefore, targeting this process may allow an abatement in vascular age acceleration to attenuate aortic stiffening, blood pressure, and end-organ damage⁸¹.

Ang II stimulates “stress-induced” premature senescence (SIPS) in VSMCs and mouse aorta, leading to a permanent cell cycle arrest through induction of cyclin dependent kinase inhibitors (CDKi) p16^INK4A and p53/p21^{CIP1 78, 81–83}. Senescence in circulating endothelial progenitor cells is increased in SHR, DOCA-salt treated rats and hypertensive humans⁸⁴. Expression of p16^INK4A is increased in heart, kidney and vasculature of DOCA-salt hypertensive rats, which was attenuated by antihypertensive medications⁸⁵. Arterial telomere uncapping and p21 activity are linked to hypertension independently of mean telomere length in humans⁸⁶.

Senescent cells evade immune clearance and undergo senescence-associated heterochromatin foci changes, leading to upregulation of anti-apoptotic proteins⁸⁷. This dictates their ability to reside in tissues for long periods and contribute to organismal dysfunction^{87, 88}. Upregulation of NF-κB signaling occurs in senescent cells and coincides with SASP, with increased secretion of inflammatory cytokines^{89, 90}. SASP has been shown to contribute to frailty and inflamm-aging in other cell systems⁹⁰. Removing senescent cells via a senolytic drug or genetic clearance reduced atheroma formation and maturation in LDL receptor knockout mice⁷⁵ and improved vascular relaxation in aged arteries via VSMC sensitization to NO⁹¹. At the cellular level, removal of Ang II-induced senescent endothelial cells via a senolytic treatment prevented enhanced leukocyte adhesion⁹². While findings are still limited regarding the role of senescence and SASP in hypertension⁸¹, these findings support the contribution of senescence and SASP to human hypertension.

ER Stress Modulation of Vascular Senescence

ER proteostasis mechanisms decline with chronological age⁹³ and aging is a risk factor for hypertension and vascular stiffening^{4, 44}. ER stress and subsequent UPR induction are associated with all major senescent hallmarks like increased CDKi and β galactosidase expression⁹⁴. A relationship between ER stress and senescence has been identified in vascular disease. However, the ability to reduce senescent cell accumulation via pharmacological reduction of ER stress remains unclear. In apolipoprotein E^−/− mice, VSMCs overexpression of senescence-associated progerin exacerbated atherosclerosis via increased ER stress markers, including GRP78, in the medial layer. Pharmacological inhibition of ER stress via TUDCA ameliorated aortic adventitia-to-media thickness, lipid deposition, and improved survival rate in these mice⁹⁵. In aged mice, the ER resident chaperone protein involved in Ca²⁺ handling, calreticulin, is significantly decreased in mesenteric arteries. Calreticulin knockdown impairs Ca²⁺ signals and endothelial-dependent vasodilation which can be restored with TUDCA treatment in aged mice⁹⁶. NOX4 gene expression, as well as expression of genes involved in ER stress, was significantly increased in aortas from aged mice. In addition, NOX4-derived ROS controlled endothelial eNOS uncoupling, which was dependent on IRE1α oxidation by sulfonylation⁹⁷. As mentioned, at the cellular level, 4-PBA treatment prevented Ang II-induced senescence in VSMCs²⁶ as well as ECs⁹². This represents a novel target since aging induces post-translational modifications of UPR signaling molecules and contributes to vascular dysfunction.

Mitochondrial Dysfunction and Vascular Aging

Reduced mitochondrial biogenesis, reduced mitophagy, increased mtDNA damage, increased mitochondrial ROS with reduced antioxidant defenses are shared hallmarks of both aged and hypertensive vessels⁹⁸. Oral supplementation with a mitochondrial antioxidant, MitoQ, has been shown to increase NO-mediated endothelial function by 42% over placebo conditions in older adults⁹⁹. Stress signaling effects of heat shock binding partner-1 (HSB-1) inhibition are mediated via increased histone H4 levels that results in enhanced compaction of both nuclear and mitochondrial genomes. Histone levels decline with age resulting in increased genomic instability, and therefore increased H4 reduce mtDNA damage while simultaneously reducing ETC complex transcription to mitigate excess age-associated mitochondrial oxidative stress¹⁰⁰. Senescent cells have hyperfused mitochondria that collect damage. Therefore, enhancing mitophagy clearance mechanisms may reduce vascular senescence. Loss of DRP1 during senescence exacerbates ECs dysfunction by increasing mitochondrial ROS and subsequently inhibiting autophagic flux¹⁰¹. However, in another study inhibition of DRP1 activity via mdiv1 reduces abdominal aortic senescence⁴⁸.

Responses Against Oxidative Stress

Disruption of the fine balance between free radicals generation and removal is an important cause of cellular stress^102–105. Many ROS, including O₂^.−, H₂O₂, hydroxyl radical (OH⁻), peroxynitrite (ONOO⁻), are highly relevant in oxidative processes that lead to protein inactivation or degradation, reduced NO bioavailability, activation of inflammatory and apoptotic cellular responses^{105, 106}. NADPH oxidase, cyclooxygenases, uncoupled eNOS, xanthine oxidase, and mitochondrial ETC directly contribute to unbalanced redox processes and arterial damage^{17, 106}. Antioxidant defenses are critical in redox-related stresses to prevent ROS-induced oxidative damage and to restore cellular redox homeostasis¹⁰⁷.

The Antioxidant Defense Pathway

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) - Kelch-like ECH-associated protein-1 (KEAP1), considered “the” cellular homeostatic guardian, stimulates expression of many antioxidant enzymes. NRF2, encoded by the NFE2L2 gene, is a stress-responsive transcription factor that belongs to cap’n’collar (CNC) subfamily of basic-leucine-zipper (bZIP) transcription factors¹⁰⁸. The protective effects of NRF2 against environmental insults was noted when Itoh and colleagues¹⁰⁹ observed that electrophile counterattack response is abrogated in NFE2L2-null mice. The electrophile counterattack response is a crucial cellular adaptation¹⁰⁸ that protects sulfhydryl groups from oxidation, reduction, or alkylation - events that lead to cell functional loss or death. To activate this response, sulfhydryl reactive electrophiles in specific molecular sensors evoke a coordinated increase in phase II enzymes, such as glutathione S-transferases, epoxide hydratase, AD(P)H: quinone oxidoreductase, and other detoxication enzymes^{108, 110}. NRF2 is the trans-regulatory factor that interacts with the antioxidant responsive element (ARE) and increases the expression of these enzymes^{108, 111, 112}.

The isolation and characterization of KEAP1 clarified how the cell senses and appropriately responds to oxidative attacks¹¹³. KEAP1 is a thiol-rich protein that promotes NRF2 degradation in unstressed conditions. Pro-oxidative stimuli directly modify KEAP1 thiols, resulting in KEAP1 inactivation, NRF2 stabilization and induction of cytoprotective genes via ARE^{114, 115}. KEAP1 is therefore the direct biosensor for electrophiles and ROS unbalance, while NRF2 is the molecular effector that maintains cellular redox homeostasis.

NRF2 Pathway, Hypertension and CVD

Pharmacological activation of NRF2 by tert-butylhydroquinone prevents Ang II-induced hypertension, microvascular remodeling, and endothelial dysfunction in wild-type, but not in Nrf2-deficient mice¹¹⁶. The importance of NRF2 activation in hypertension-associated vascular dysfunction is further supported by studies showing that SHRSP have reduced NRF2 activity and NRF2-targeted gene expression, events linked to the vascular dysfunction exhibited by these animals. NRF2 activators (bardoxolone/sulforaphane) improve SHRSP’s vascular function and reduce Ang II-induced ROS generation in VSMCs¹¹⁷. NRF2 activation by sulforaphane maintains a healthy endothelial phenotype under proinflammatory conditions¹¹⁸, whereas disruption of Nrf2 signaling impairs angiogenic responses in ECs¹¹⁹. In two kidney-one clip (2K-1C) rats, oral nitrite administration increases NRF2 activation and expression of NRF2-regulated genes (SOD1, catalase, glutathione peroxidase, thioredoxin-1 and −2) and attenuates vascular dysfunction¹²⁰, suggesting that NRF2 activation contributes to the antioxidant effects of oral nitrite.

Selective Nrf2 gene deletion in the rostral ventrolateral medulla (RVLM) increases sympathetic outflow and impairs baroreflex function, inducing hypertension, potentially by disrupting antioxidant enzyme expression¹²¹. Of clinical importance, single nucleotide polymorphism in NFE2L2 (rs6721961 CA+AA) and KEAP1 (rs110857735 the AA or CA) is linked to altered blood pressure and adverse cardiovascular outcomes in Japanese¹²² and Italian¹²³ patients, respectively.

Interestingly, Ang II-infused Nrf2^−/− mice exhibit exaggerated cardiac hypertrophy with no difference in the development of hypertension compared to wild-type mice¹²⁴. Despite evidence supporting an anti-hypertensive role for NRF2 activation, enhancement of NRF2 activity via genetic inhibition of KEAP1 appears rather harmful, since it leads to higher blood pressure upon Ang II infusion¹²⁵.

NRF2 also prevents vascular damage in diabetes, a condition that increases the risk of hypertension. Diabetic db/db mice exhibit decreased renal NRF2 activity, downregulation of antioxidant enzymes and nephropathy, events that are mediated the adipokine chemerin and its receptor ChemR23¹²⁶. Impaired NRF2 signaling can be associated with disrupted Akt-dependent signaling¹²⁷. Although KEAP1 is key to regulate NRF2 activity in response to electrophiles, NRF2 is also activated via PI3K-AKT signaling¹²⁸. Akt induces NRF2 activity¹²⁹ primarily by inhibiting GSK-3, glycogen synthase kinase-3β, which phosphorylates NRF2 and marks it to ubiquitination and degradation¹³⁰. In streptozotocin-induced diabetic mice, Nrf2 activation by L-sulforaphane or bardoxolone rescues internal pudendal artery function¹³¹, suggesting that this antioxidant system may be important in numerous vascular dysfunction conditions, as shown in Table 1. In this context, understanding NFR2 signaling in physiological and pathological conditions not only clarifies its function in redox balance, but also contributes to unravel potential therapeutic strategies targeting vascular dysfunction.

NRF2 in Proteostasis and Senescence

NRF2 not only protects from oxidative stress, but also modulates general adaptive cellular processes, such as inflammation¹³², metabolic programming¹³³ and proteostasis¹³⁴. In vascular stress, NRF2 is activated via PERK phosphorylation, as a protective mechanism to limit CHOP-induced apoptosis in ECs¹³⁵. NRF2 pharmacological inhibition with ML385, in a myocardial infarction mouse model, suppresses cardioprotective effects of tumor susceptibility gene 101 (Tsg101), which promotes p62 aggregation, KEAP1 degradation and NRF2 translocation to the nucleus¹³⁶. This highlights a link between proteostasis and NRF2 antioxidant responses. In senescent vascular cells, the lack of an effective NRF2 response to oxidative stress results in overwhelming ROS levels^{137, 138}. Nrf2 null mice have higher levels of IL-1β and TNF-α, well-known components of the SASP. Additionally, Nrf2 deletion increases the expression of molecular senescence markers (p16INK4a, p21) in cerebral arteries of 24-month-old mice¹³⁹. Endothelial Nrf2 mRNA is reduced by miRNAs derived from senescent cells, such as miR-126, miR-21, and miR-100¹⁴⁰, further reinforcing that NRF2 is also a potential therapeutic target to treat aging-related diseases¹⁴¹. Figure 3 illustrates how NRF2 modulates vascular (dys)function.

Figure 3. NRF2 and oxidative stress.

Impaired NRF2 activation reduces the expression of antioxidant proteins, leading to exacerbation of cellular oxidative damage. In endothelial cells, ROS overproduction reduces NO bioavailability, impairs vasodilation, causes inflammatory endothelial activation, ER stress and mitochondrial dysfunction. In VSMCs, oxidative damage also activates proliferative pathways and increases vasoconstrictor responses. NADPH Oxidase (NOX), Toll-like receptors (TLR), angiotensin II (Ang II), nitric oxide (NO), nuclear factor (erythroid-derived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), antioxidant response element (ARE), Small Maf proteins (sMAF), damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs). Created with BioRender.com.

NRF2-inducers/activators, such as bardoxolone (government identifier: NCT00811889; NCT01351675)¹⁴², and sulforaphane (government identifier: NCT01008826; NCT02880462; NCT02801448; NCT03220542)¹⁴³ are currently being tested in clinical trials. Other natural NRF2 activators, such as polyphenols, phytosterols, and terpenoids (e.g., Baicalein), are also being tested in pre-clinical models of kidney disease¹⁴⁴. So far, data from these studies suggest that modulation of the NRF2-KEAP1 pathway is a potential therapeutic strategy to combat oxidative stress-induced cellular damage. This raises the possibility of successful results also in CVD. However, contrasting results have been reported. While bardoxolone improves estimated glomerular filtration rate in chronic kidney disease patients¹⁴² and sulforaphane improves glucose metabolism in patients with dysregulated type 2 diabetes¹⁴³, bardoxolone failed to show positive effects in patients with type 2 diabetes and end-stage chronic kidney disease¹⁴⁵. The negative or unsuccessful result in this specific study may be linked to the high-risk group of patients enrolled in the trial.

Although countless preclinical studies show detrimental roles of ROS and oxidative stress in the cardiovascular system as well as beneficial cardiovascular effects of antioxidants, many clinical trials with antioxidant compounds [e.g. with β-carotene, vitamin E, vitamin C, α-lipoic acid, polyphenols (epigallocatechin gallate, curcumin, resveratrol) and flavonoids (quercetin, rutin)] have not confirmed beneficial or protective effects¹⁴⁴. In many cases, adverse effects such as cardiac ischemia, intracerebral hemorrhage, increased risk of heart failure, decreased vasodilation, and oxidative damage, were reported. Adverse effects are usually observed with high doses of antioxidants and many studies show that antioxidants exhibit dose-dependent effects¹⁰². Potential possibilities to explain the negative results in the clinical trials include: i) antioxidant drugs may abrogate physiological and relevant oxidant processes; ii) antioxidants reduce metal ions, such as Fe³⁺ and Cu²⁺, favoring the formation of other free radicals (•OH, e.g.); iii) antioxidants may not distinguish between radicals involved in physiological processes and those that cause oxidative damage; iv) removal of oxidants may disrupt essential endogenous antioxidant responses (e.g. NRF2 signaling) and thus decrease endogenous antioxidant protection. These hypotheses led to terms such as reductive stress, antioxidative stress and antioxidant paradox. Additional possibilities include v) antioxidants may modify macromolecules, which by unrelated mechanisms negatively impact beneficial effects; vi) antioxidant effects may be hampered by inadequate duration of treatment, poor absorption and/or weak scavenging activity. The beneficial vascular effects of physiological ROS and how excessive removal of ROS might perturb vascular homeostasis was recently reviewed¹⁰².

Many authors argue that antioxidants should be target-specific – i.e. directed to selective inhibition of ROS-generating enzymes (e.g. NADPH oxidase isoforms) or to activation of specific antioxidant signaling (such as NRF2 and specific antioxidant enzymes)¹⁴⁶. Others consider that antioxidants should be given in low doses in order to avoid suppression of physiological ROS; or that administration of low levels of pro-oxidants may enhance the antioxidant capacity of cells^{102, 146}. Finally, inclusion of specific groups of patients in the clinical trials should be considered, since some of the negative results were obtained in very ill patients, including patients with end-stage chronic kidney disease plus type 2 diabetes¹⁴⁵. Studies on all these topics are still incipient but may reveal new strategies against the detrimental effects of oxidative stress in hypertension and CVD.

Signaling Via Pattern Recognition Receptors

Pattern recognition receptors (PRRs), i.e. proteins found in the cell membrane, in subcellular compartments, in the cytosol, and extracellularly, recognize molecules expressed by pathogens (the so-called pathogen-associated molecular patterns – PAMPs) or molecules released by damaged cells (the damage-associated molecular patterns – DAMPs). Four major sub-families of PRRs are known: Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)- Leucine Rich Repeats (LRR)-containing receptors (NLR), retinoic acid-inducible gene 1 (RIG-1)-like receptors (RLR), and C-type lectin receptors (CLR). PRRs not only activate pro-inflammatory responses, but also activate and regulate cell death signaling pathways^147–149. TLR, for instance, can trigger apoptosis, while inflammasomes can activate key proteins in necroptotic cells, as well as caspase-11, important for pyroptosis¹⁵⁰. Of note, in a clinical trial for patients with refractory solid tumors, the use of a vaccine containing NOD2 and TLR9 stimulants worsened arterial hypertension from grade 2 to grade 3 in 20% of the patients, indicating their role on rising blood pressure¹⁵¹. Thus, due to solid evidence linking TLRs and NLRP3 inflammasome to arterial hypertension and CVD, in this last section these critical PRRs will be discussed.

Toll-Like Receptors

The discovery of TLRs as PRRs changed the idea that the innate immune system is a simple mechanism to initiate the adaptative immune response, shedding light for a broader understanding of the innate immune system in health and disease¹⁵². TLRs are expressed in immune and nonimmune cells. In the cardiovascular system, TLRs expressed in ECs and VSMCs sense the danger in the advent of a pathogen invasion to trigger the inflammatory response and control the local blood flow ¹⁵³. As shown in Figure 4, TLRs are also activated by DAMPs, which are fragments of damaged or dying cells that could cause a chronic low-grade inflammation by prolonged or excessive activation of the TLRs, causing endothelial dysfunction and CVD¹⁵⁴.

Figure 4. Ligands for TLRs and NLRP3 inflammasome.

TLRs and NLRP3 inflammasome are involved in a growing number of infectious, autoimmune, and metabolic diseases. Nearly every class of microbe and many cell-derived as well as synthetic compounds activate or modulate TLRs and NLRP3 inflammasome. The Hemozoin, disposal product formed from the digestion of blood by some blood-feeding parasites such as Plasmodium. Nigericin, microbial toxin derived from Streptomyces hygroscopicus. Flagellin, structural component of the bacterial flagellum. PFT, pore-forming toxins. ATP, adenosine 5’-triphosphate. Drusen, aging- and macular degeneration-associated accumulations of extracellular material that build up between Bruch’s membrane and the retinal pigment epithelium of the eye. Nano-SiO₂, silica dioxide nanoparticles.

At least 10 functional TLRs have been identified in human cells. TLR1, TLR2, TLR5, TLR6 and TLR10 are expressed in the plasma membrane of the cells, while TLR3, TLR7, TLR8, TLR9 are expressed in the endosomes and ER and, lastly, TLR4 can be found in both plasma membrane and endosomal vesicles¹⁵⁵. In rodents, TLR11, TLR12 and TLR13 are also described^{156, 157}. Nonetheless, TLR activation is mediated by specific ligands and the downstream pathway of TLRs is associated with different adaptor proteins and ultimately leading to the production of proinflammatory cytokines and interferons. The role of TLRs in hypertension is not fully elucidated. However, there is evidence of the contribution of TLRs for endothelial and vascular dysfunction leading to hypertension. Figure 5 illustrates the downstream pathway in TLR activation.

Figure 5. Activation of TLRs pathway and their downstream targets.

In hypertension, TLRs expressed in the cell membrane or in the endosomes are activated by damage-associated molecular patterns (DAMPs) derived from the cellular damage and it triggers the downstream signaling pathway through the activation of adaptor molecules myeloid differentiation protein (MyD88) and TIR domain-containing adaptor protein inducing interferon-β (TRIF) to induce the activation of NF-κB, which will, ultimately, lead to the formation of proinflammatory cytokines and interferons.​

Among the TLRs, activation of TLR2 (which is known for heterodimerizing with TLR1 or TLR6 to recognize triacyl or diacyl lipopeptides, respectively) was shown to play a role in the regulation of the vascular tone. Specifically, vessels exposed to pathogens exhibited a diminished contractile force, which was not observed in TLR2^−/− mice. Activation of TLR2 is associated with inducible nitric oxide synthase (iNOS)-derived NO formation, in a mechanism dependent on heterodimerization of TLR2 with TLR6 and subsequent activation of the adaptor protein MyD88 (myeloid differentiation protein), MAL (MyD-adapter-like protein) and TNF-α production¹⁵⁸. In addition, treatment with Ang II led to the development of fibrosis in the aorta as well as endothelial to mesenchymal transition (EndMT) via NF-κB signaling in human umbilical vein ECs, through activation of TLR2, and these effects were prevented by knocking out or silencing TLR2¹⁵⁹. Although activation of TLR2 by pathogens led to an anticontractile effect on the vessels, damage-associated activation of TLR2 seems to contribute to endothelial dysfunction and the development of hypertension. Increased systolic blood pressure (SBP) was observed in mice injected with abnormal HDL containing symmetric dimethylarginase (HDL+SDMA) obtained from patients with chronic kidney disease (CKD). However, injection of HDL+SDMA in TLR2^−/− mice (but not in TLR4^−/−) failed to increase SBP, supporting the idea that the increase in SBP was mediated by activation of TLR2¹⁶⁰. In ECs, HDL+SDMA decreased NO bioavailability and increased ROS, however, by a mechanism independent of TLR1 or TLR6 dimerization¹⁶⁰. In addition, in vascular smooth muscle, dysfunctional HDL from mice with CKD or humans with end-stage renal disease induced the mRNA expression of MCP-1 (monocyte chemoattractant protein 1) through activation of TLR2 and TLR4¹⁶¹.

Indeed, evidence has shown that TLR4 also plays a role in endothelial and vascular dysfunction in several pathological conditions, including diabetes¹⁶² and sepsis¹⁶³. Classically, TLR4 is the specific sensor for lipopolysaccharide and its activation leads to the recruitment of the adaptor protein MyD88/MAL or TRIF (TIR domain-containing adaptor protein inducing interferon-β; TIR: Toll/IL-receptor domain), to produce several proinflammatory cytokines or interferons, respectively¹⁶⁴. In addition, high mobility group box 1 (HMGB1), an alarmin and well recognized as a member in SASP, has been highly associated with the activation of TLR4 and receptor for advanced glycation end-products (RAGEs). HMGB1 and TLR4 activation is correlated with pulmonary hypertension¹⁶⁵ and preeclampsia¹⁶⁶. Activation of TLR4 also plays a role in the development of vasculogenic erectile dysfunction, which is often considered an early marker for CVD¹⁶⁷. Although TLR4 is less investigated in arterial hypertension, administration of VIPER (a specific TLR4 blocker) in the paraventricular nucleus (PVN) of SHR for 14 days, reduced mean arterial pressure (MAP) and expression of TLR4 in the PVN and plasma. Additionally, VIPER reduced PVN levels of HMGB1, and decreased expression of TNF-α, IL-1β, iNOS while increasing expression of IL-10¹⁶⁸. Similarly, in two-kidney, one-clip (2K1C) hypertensive rats, physical exercise attenuated the increase in SBP and reduced expression of TLR4, and its downstream effectors MyD88, NF-κB, TNF-α and IL-1β in the PVN with the same magnitude as the TLR4 inhibitor TAK242¹⁶⁹. In addition, SHR treated with losartan or an herbal product displayed reduced expression of TLR4, MyD88, NF-κB, TNF-α and IL-6, which was accompanied by an improvement in cardiovascular parameters (SBP, DBP, MAP, HR and flow), prevention of cardiac hypertrophy and fibrosis and preserved endothelium-dependent vascular relaxation¹⁷⁰. Similarly, SHR exhibited increased expression of TLR4 in mesenteric arteries and treatment with anti-TLR4 decreased blood pressure and it reduced the expression of TLR4, cyclooxygenase-2 (COX-2) and thromboxane A2. The contractile response to norepinephrine in the mesenteric artery, as well as the circulating levels of IL-6¹⁷¹ were also normalized after treatment with anti-TLR4. In humans, HMGB-1 is increased in the plasma of patients with peripheral artery disease and it is positively correlated with the severity of PAD¹⁷². Furthermore, endothelial dysfunction is associated with increased levels of HMGB1 in women with polycystic ovary syndrome¹⁷³. Despite the correlation of TLR4 with vascular complications in different diseases and contrary to what has been observed in SHR, Ang II infusion in TLR4^−/− mice did not prevent the increase in the SBP, although the cardiac hypertrophy was attenuated¹⁷⁴. Thus, the role of TLR4 in hypertension is still controversial. On the other hand, infusion of Ang II in TLR3^−/− mice prevented the increase in the SBP as well as the cardiac hypertrophy¹⁷⁴. TLR3 is localized in the endosomes and senses viral nucleic acids or mitochondrial double-stranded RNA released from damaged cells. Its downstream signaling pathway occurs exclusively through TRIF¹⁷⁵. Interestingly, many viral infections are related to increased blood pressure or hypertension, therefore, activation of TLR3 could be the missing link between these conditions⁵. Corroborating this idea, TLR3 activation is associated with endothelial dysfunction and increased ROS¹⁷⁶ and it is also correlated with increased blood pressure in animal models of preeclampsia¹⁷⁷. Activation of TLR3 in human microvascular ECs causes augmented expression of pro-inflammatory cytokines and damages to the endothelial barrier integrity, which are potentiated when eNOS is silenced or inhibited with L-NAME¹⁷⁸. Although the inflammation caused by activation of TLR3 does not play a role in the development of arterial hypertension, it is possible that TLR3 activation worsens the endothelial function in hypertension, by decreased expression/activity of eNOS.

Similar to TLR3, TLR9 is expressed in endosomes of immune and nonimmune cells, and it is activated by unmethylated DNA. Upon activation of TLR9, the adaptor protein MyD88 forms a complex with IL-1-receptor-activated kinase (IRAK4) and TNF receptor-associated factor (TRAF6), to induce the activation of NF-κB and generate proinflammatory cytokines¹⁵⁵. In SHR, impaired autophagic machinery led to increased circulating levels of mtDNA, activating TLR9 and leading to vascular dysfunction¹⁷⁹ whereas inhibition of TLR9 in young SHR (but not in adult SHR) led to a milder increase in blood pressure and recruitment of immune cells to the vessels when compared to young SHR with intact TLR9¹⁸⁰. In addition, adult male SHR exhibit higher SBP compared to female SHR, and likewise, circulating levels of mtDNA and pro-inflammatory cytokines, as well as contractile responses to phenylephrine are higher in male SHR than in female SHR¹⁸¹, reinforcing the idea that the increase in blood pressure is mediated by TLR9 activation. Furthermore, endothelial/vascular dysfunction associated with TLR9 activation is also observed in other pathological conditions such as prediabetes¹⁸², preeclampsia¹⁸³ and atherosclerosis¹⁸⁴.

Other TLRs have been reported to contribute to endothelial and/or vascular dysfunction in some cardiovascular complications/diseases, however, not in arterial hypertension. It is known that TLR7 and TLR8 activate downstream signaling pathways similarly to TLR9, through NF-κB release and formation of the proinflammatory cytokines TNF-α, IL-6, IL-1β, IL-12 and type 1 interferon¹⁸⁵. The implication of TLR7 and TLR8 for CVD are not well established. However, a recent study showed that, in mice, the topical administration of imiquimod to induce lupus through TLR7 activation also induced hypertension, endothelial dysfunction, decreased eNOS expression and caused vascular remodeling, associated with increased levels of proinflammatory cytokines¹⁸⁶. In contrast, another study showed that the activation of TLR7 (as well as TLR3 and TLR8) leads to an increase in blood pressure and endothelial dysfunction only in pregnant mice and it was also found that the levels of these TLRs were increased in the placenta of women with preeclampsia¹⁸⁷. Together, this can be indicative that the activation of TLR7 (and 8) does not trigger the development of vascular complications, unless upon the existence of another inflammatory condition. As for TLR8, its activation seems to be involved in the development of atherosclerotic plaques in rabbits fed high lipid diet¹⁸⁸. In addition to the inflammatory state observed by overactivation of the TLRs in these pathological conditions, further mechanisms have been demonstrated that could link TLRs and hypertension.

RhoA/Rho-kinase (ROCK) signaling is critical in arterial contraction and hypertension. ROCK negatively regulates the myosin light chain phosphatase subunit, myosin phosphatase target subunit 1 (MYPT1), a physiological ROCK substrate. Thus, ROCK activation causes Ca²⁺ sensitization in VSMCs, favoring the contractile state, and its overexpression/activity is largely associated with hypertension¹⁸⁹. Interestingly, studies have reported a communication between some TLRs and activation of RhoA/ROCK signaling in the vasculature and other organs, such as in the corpus cavernosum, where activation of TLR1/2 increases contraction and impairs relaxation to Y-27632, suggesting that TLR1/2 activation leads to increased ROCK signaling¹⁹⁰. Similarly, a noncanonical pathway related to the activation of TLR9 has been described and it could be involved in the underlying mechanism of TLR9-induced hypertension. In VSMCs, treatment with the TLR9 agonist ODN2395, induced vascular autophagy by activation of the AMPK pathway via TGF-β–activated kinase 1 (TAK1), as well as phosphorylation of MYPT1 and cofilin, leading to actin polymerization and impaired arterial relaxation¹⁹¹. Interestingly, restoration of arterial autophagy in SHR improved remodeling and endothelium-dependent relaxation of the mesenteric resistance artery and reduced the expression of ROCK and phosphorylation of MYPT-1, preventing the early vascular aging in hypertension⁴⁴. Overall, activation of different TLRs can trigger mechanisms associated with the development of hypertension (Table 1). Nevertheless, TLRs are not the only PRRs that may be involved in this process.

NLRP3 Inflammasome

Certain members of the PRRs are assembled in complex structures or platforms called inflammasomes. Canonical inflammasomes are composed of a sensor protein, usually a NLR or a pyrin and HIN domain-containing protein (PYHIN), an adaptor molecule, the apoptosis-associated speck-like protein containing a caspase activating and recruitment domain (ASC), and pro-caspase-1. Activation of the cytosolic sensors in response to cell stress [represented by PAMPs, DAMPs or cytosolic instabilities (ionic imbalance, oxidative stress)] leads to the recruitment and activation of caspase-1 either directly or through the ASC adaptor molecule. Among the five major types of inflammasomes [NLR pyrin domain-containing 3 (NLRP3), NLRP1, neuronal apoptosis inhibitory protein (NAIP)/NLR CARD-containing 4 (NLRC4), absent in melanoma 2 (AIM2), and PYRIN], NLRP3 has been linked to uncontrolled inflammation in many CVD, including arterial hypertension^{192, 193}.

NLRP3 inflammasome activation involves two coordinated steps, as shown in Figure 6. In the first step (priming step), DAMPs or PAMPs-induced activation of TLRs lead to NF-kB signaling, synthesis of pro-IL-1β, pro-IL-18 and increased expression of NLRP3 components. The second step involves NLRP3 inflammasome assembly, which triggers the self-cleavage and activation of caspase-1, and conversion of pro-IL-1β and pro-IL-18 to their mature forms¹⁹⁴. An intriguing and not yet resolved point refers to the mechanisms linking the priming and activation steps. Distinct stimuli can trigger this process, including pathogens, K⁺ efflux, increased intracellular Ca²⁺, lysosomal leakage, mitochondrial damage and ROS (derived from ER stress, damaged mitochondria and NADPH oxidase)^194–196. ER stress activates NF-κB, thioredoxin-interacting protein (TXNIP) and sterol regulatory-element binding proteins (SREBPs) signaling, Ca²⁺ release, and ROS production. However, a common intracellular intermediate that leads to NLRP3 assembly and activation has not been identified. Several kinases and phosphatases modify NLRP3 in response to either PAMPs or DAMPs and provide fine adjustments of NLRP3 activation^{150, 197}.

Figure 6. Two step model for NLRP3 inflammasome activation.

In hypertension and aging, the priming signal (first signal) is provided by proinflammatory cytokines, such as IL-1β and TNF, or molecules released from damaged cells (the so called damage-associated molecular patterns – DAMPs) that interact with specify membrane receptors (IL-1R, TNFR, TLR) leading to activation of transcription factor nuclear factor-kappa B signaling. NF-κB upregulates gene expression of NLRP3 components (NLRP3, ASC adaptor molecule), pro-caspase 1, pro-IL-1β, pro-IL-18 and other pro-inflammatory mediators. The complete NLRP3 assembly is induced by a second signal, that can be represented by a plethora of factors - K+ efflux, increased intracellular Ca²⁺, lysosomal leakage, mitochondrial damage and ROS mainly derived from ER stress, damaged mitochondria [(mt)ROS and mtDNA], and NADPH oxidase (NOX). ER stress also activates NF-κB, TXNIP, and SREBP signaling, further contributing to NLRP3 activation. Activated NLRP3 recruits and activates caspase-1 that cleavages pro-IL-1β, pro-IL-18 and FL-GSDMD (Gasdermin D) to their respective active forms. GSDMD induces pyroptotic cell death and IL-1β and IL-18 contribute to hypertension- and aging-associated vascular damage. Created with BioRender.com.

IL-1β and IL-18 receptors (IL-1R and IL-18Rα, respectively), are found on several leukocyte subsets, ECs and VSMCs^{198, 199}. Activation of IL-1R and IL-18Rα leads to recruitment of distinct accessory and adapter molecules that facilitate the activation of JNK and p38 MAPK signaling and transcription factors such as activator protein-1 (AP-1) and NF-κB²⁰⁰. These receptors and factors stimulate pro-inflammatory gene expression^{201, 202} and are relevant to hypertension-associated vascular dysfunction^{203, 204} and remodeling^{201, 205–207}. In a feedback loop, IL-1β /IL-18 signaling stimulates ROS production by NADPH oxidase²⁰⁶ and COX-dependent mechanisms²⁰⁴ and, consequently, induces NLRP3 overactivation.

Overactivation of the NLRP3 inflammasome has been linked to the development of arterial hypertension and CVD. Omi et al., by examining 1911 patients (987 with hypertension, 924 controls), reported that homozygotes of 12 repeat allele, whose NLRP3 inflammasomes produce more chemokines after stimulation, exhibit greater risk for the development of hypertension compared to heterozygotes or homozygotes of non-12 repeat allele (who exhibit low NLRP3 activity). In addition, mean systolic blood pressure of male homozygotes of 12 repeat allele (high NLRP3 activity) was 6.4 mmHg higher than that of homozygotes of low NLRP3 activity alleles²⁰⁸. Furthermore, hypertensive patients, with hyperaldosteronism and with normal aldosterone levels, exhibit increased NLRP3 inflammasome activation²⁰⁹.

IL-1β and IL-18, end-products of NLRP3 activation, are increased in hypertensive patients^{205, 210}. A recent clinical trial (Canakinumab Anti-inflammatory Thrombosis Outcome Study – CANTOS) showed that canakinumab, a human monoclonal antibody targeting IL-1β, reduces rates of recurrent cardiovascular events^{211, 212} and hospitalization for heart failure²¹³ in patients with a history of myocardial infarction and persistent pro-inflammatory response. In this context, treatment of DOCA/salt-hypertensive mice with anakinra, an IL-1R antagonist, reduces blood pressure and renal fibrosis, although it does not change kidney leukocyte infiltration²¹⁴. Although it is clear that IL-1β, the main effector of NLRP3, is important in the development of CVD, including arterial hypertension, the complex mechanisms involved in IL-1β effects on hypertension need further investigation.

NLRP3 blockade also prevents hypertension-associated cardiovascular dysfunction, oxidative stress and structural abnormalities. NLRP3 gene silencing attenuates hypertension, vascular remodeling, and VSMCs phenotype switching in SHR¹⁹³. Nlrp3 deletion in mice prevents Ang II-induced gestational hypertension and IL-6 up-regulation in the placenta and kidneys²¹⁵. Additionally, the NLRP3 inflammasome, through activation of IL-1R, mediates aldosterone-induced vascular damage²⁰⁹ and MCC950, a selective NLRP3 inhibitor²¹⁶, reduces blood pressure and attenuates renal damage and dysfunction in salt-sensitive hypertension¹⁹². Of note, MCC950 failed to improve vascular function in aged mice with Ang II-induced hypertension²¹⁷. Salt-sensitive hypertensive rats exhibit increased IL-1β and NLRP3 in the hypothalamic paraventricular nucleus (PVN). Bilateral injection of gevokizumab (IL-1β inhibitor) into the PVN decreased mean arterial pressure and heart rate in these animals, suggesting that NLRP3 / IL-1β signaling contributes to sympathoexcitation during development of salt-dependent hypertension²¹⁸.

NLRP3 inflammasome also contributes to vascular damage in diseases closely linked to hypertension, such as atherosclerosis and diabetes. In these conditions, activation of the NLRP3 inflammasome mediates pyroptosis, increases mitochondrial ROS in ECs²¹⁹ and induces endothelial dysfunction²²⁰. NLRP3 inhibitors, such as tranilast and oridin, display anti-atherosclerotic properties by decreasing vascular and endothelial inflammation, restraining activation of MAPK and NF-κB signaling and decreasing cell migration and angiogenesis^221–223. Blockade of NLRP3 activation also restores EC function and reduces oxidative stress and inflammatory processes in type 2²²⁴ and type 1²²⁵ diabetes. In addition, NLRP3 inflammasome is linked to aging-associated dysregulation of the immune system²²⁶, endothelial senescence²²⁷, and cardiovascular remodeling and dysfunction²²⁶, ²²⁸. NLRP3 components gene expression is increased in peripheral blood mononuclear cells of older patients with vascular diseases in comparison to younger patients²²⁹.

Overall, these studies provide evidence that NLRP3 inflammasome activation contributes to proinflammatory signaling, leading to vascular dysfunction and remodeling and a premature senescence phenotype in hypertension and other CVD. The impact of genetic and pharmacological control of NLRP3 activity on vascular (dys)function is summarized in Table 1. Since NLRP3 assembly and signaling involve many molecules, NLRP3 inflammasome inhibition can be achieved by interfering with upstream molecules (P2X7 receptor, K⁺ channels, ROS), inflammasome components (NLRP3 domains - e.g. NLRP3 ATP-binding domain -, ASC), and downstream molecules (caspase-1, IL-1β and IL-18 and their receptors), as reviewed by other authors^{216, 223}.

Many questions remain unanswered, demanding further studies: i) which molecules function as cellular stress-signals to trigger NLRP3 activation in hypertension?, ii) what specific signaling pathways are activated when NLRP3 senses different cellular stress signals?, iii) how modulators of cellular redox status (pro- and anti-oxidant enzymes, ER, mitochondria) influence NLRP3 activity?, iv) do current anti-hypertensive medications impact NLRP3 activation and signaling?, v) does simultaneous blockade of NLRP3 inflammasome and IL-1β/IL-18 signaling better prevent hypertension and associated complications?, vi) how does activation of other stress signaling pathways impact NLRP3 activation (and vice-versa)?, vii) do other inflammasomes, such as NLRP1, NLRC4, AIM2, and noncanonical inflammasomes also contribute to hypertension-associated vascular dysfunction?. Another aspect to be explored is that not only immune cells express NLRP3. The inflammasome is also found in myocardial, VSMCs, EC and fibroblasts. NLRP3 inflammasome activation in these cells also contributes to proinflammatory signaling, leading to vascular dysfunction and remodeling and premature senescence phenotype^{233, 234}.

Altogether, the NLRP3 inflammasome, a sensor for danger signals and one of the earliest components activated in inflammatory responses, may represent a critical checkpoint that interconnects pathophysiological events in hypertension. Effective regulation of NLRP3 may help prevent the development and aggravation of hallmark processes in arterial hypertension and CVD, especially in conditions where current available drugs are insufficient to prevent the development or complications of these diseases.

Summary and Conclusion

Vascular changes that contribute to elevated arterial blood pressure in hypertension include the following: endothelial dysfunction, increased smooth muscle contraction, remodeling of the vascular wall, inflammation and increased arterial stiffness. This review provided a synopsis of several stress signaling mechanisms that contribute to these vascular changes and appraised literature indicating that several of these abnormalities occur during the aging process. Common cellular mechanisms involved in vascular dysfunction in hypertension and aging include ER stress and accumulation of protein aggregates, oxidative injury, damage to mitochondria, DNA damage, cell senescence, fibrosis, TLR activation and inflammasome formation. Adaptive mechanisms against these stresses include the UPR, autophagy, ARE signaling, mitophagy and mitochondrial fusion, induction of STING and senolysis. There are several major gaps in our knowledge in terms of developing pharmaceuticals to target these adaptive mechanisms. As these stress responses are all critical in cell homeostasis and defense, unappreciated side effects are expected for any potential target. From a pharmacological standpoint, future studies should consider the following: establishment of controlled targeting for maladaptive stress signaling, better use of potential translational models for screening of the targeting compounds, and investigation on the impact of lifestyle choices and environmental factors on these maladaptive stress signals and their contribution to hypertension.

Nonstandard Abbreviations and Acronyms:

4-PBA

4-phenylbutyric acid

AAA

abdominal aortic aneurysm

AIM2

absent in melanoma 2

AMPK

AMP-activated protein kinase

Ang II

angiotensin II

ARE

antioxidant response

ASC

caspase activating and recruitment domain

AT1R

Ang II type 1 receptor

AT2R

Ang II type 2 receptor

ATAAD

ascending thoracic aortic aneurysm and dissection

ATF6

activating transcription factor 6

ATM

ataxia telangiectasia mutated

ATP

adenosine triphosphate

ATP

adenosine 5’-triphosphate Nano-SiO2, silica dioxide nanoparticles

BHB

β-Hydroxybutyrate

bZIP

basic-leucine-zipper

Ca2+

calcium

CANTOS

Canakinumab Anti-inflammatory Thrombosis Outcome Study

CC

corpora cavernosa

CDKi

cyclin dependent kinase inhibitors

CHOP

C/EBP homologous protein

CKD

chronic kidney disease

CLR

C-type lectin receptors

CNC

cap’n’collar

COX-2

cyclooxygenase-2

CVD

cardiovascular diseases

DAMPs

damage-associated molecular patterns

DRP1

dynamin-related protein

ECs

endothelial cells

EndMT

endothelial to mesenchymal transition

eNOS

endothelial nitric oxide synthase

ER

endoplasmic reticulum

ERAD

ER-associated degradation

ESRD

end-stage renal disease

ETC

electron transport chain

FAM3A

family with sequence similarity 3 member A

FL-GSDMD

(Gasdermin D inactive)

GRP78

glucose-regulated protein 78

GSK-3

glycogen synthase kinase-3β

GTPases

guanosine triphosphatases

H2O2

hydrogen peroxide

HMGB1

high mobility group box 1

HO-1

heme oxygenase-1

HSB-1

heat shock binding partner-1

HSP90

heat shock protein 90

I/R

ischemia/reperfusion

IFNβ

interferon-β

IL-1β

interleukin-1β

IL-6

interleukin-6

iNOS

inducible nitric oxide synthase

IP3R

inositol 1,4,5-trisphosphate receptors

IRAK4

IL-1-receptor-activated kinase

IRE1α

inositol-requiring transmembrane kinase 1 alpha

IRF3

interferon regulatory factor 3

JNK

c-Jun N terminus kinase

K+

potassium

KEAP1

Kelch-like ECH-associated protein 1

LRR

Leucine Rich Repeats

MAL

MyD-adapter-like protein

MAMs

mitochondria-associated membranes

MAP

mean arterial pressure

MCP-1

monocyte chemoattractant protein 1

MFN1 and MFN2

mitofusins 1 and 2

miR

microRNA

MLKL

mixed lineage kinase domain-like

mtDNA

mitochondrial DNA

mtROS

mitochondrial-derived ROS

MyD88

myeloid differentiation protein

MYPT1

myosin phosphatase target subunit 1

NOX

NADPH Oxidase

NAIP

neuronal apoptosis inhibitory protein

NF-κB

nuclear factor-κB

NLR

containing receptors

NLRC4

NLR CARD-containing 4

NLRP3

NLR pyrin domain-containing 3

NO

nitric oxide

NOD

nucleotide-binding oligomerization domain

NOX

HADPH oxidase

Nrf2

nuclear factor (erythroid-derived 2)-like 2

O2.-

superoxide anion

OH-

hydroxyl radical

ONOO−

peroxynitrite

OPA1

optic atrophy 1

ox-LDL

oxidized LDL

p38 MAPK

p38 mitogen-activated protein kinases

PAMPs

pathogen-associated molecular patterns

PRRs

pattern recognition receptors

PERK

protein kinase-like ER kinase

PFT

pore-forming toxins

pro

IL-18

pro

IL-1β

PVN

paraventricular nucleus

PYHIN

pyrin and HIN domain-containing protein

RAGE

receptor for advanced glycation end-products

RhoA

Ras homolog family member A

RLR

inducible gene 1 (RIG-1)-like receptors

ROCK

RhoA/Rho-kinase

ROS

reactive oxygen species

RVLM,

rostral ventrolateral medulla

SAHF

senescence-associated heterochromatin foci

SASP

senescence-associated secretory phenotype

SBP

systolic blood pressure

SHR

spontaneously hypertensive rats

SHRSP

stroke-prone spontaneously hypertensive rats

SIRT3

mitochondrial sirtuin 3

sMAF

small Maf proteins

SODs

superoxide dismutases

STING

signaling effector stimulator of interferon genes

TAK1

transforming growth factor β–activated kinase 1

TBK1

TANK- binding kinase 1

TGF-β1

transforming growth factor-β1

TLR

Toll-like receptors

TNF-α

tumor necrosis factor-α

TRAF6

TNF receptor-associated factor

TRIF

TIR-domain-containing adapter-inducing interferon-β

TUDCA

tauroursodeoxycholic acid

TXNIP

thioredoxin-interacting protein

UPR

unfolded protein responses

VCP

valosin-containing protein

VDAC1

voltage-dependent anion-selective channel protein

VSMCs

vascular smooth muscle cells

XBP-1s

X-box-binding-protein 1 spliced isoform