Mechanisms of pulmonary vascular dysfunction in pulmonary hypertension and implications for novel therapies

Abstract

Pulmonary hypertension (PH) is a serious disease characterized by various degrees of pulmonary vasoconstriction and progressive fibroproliferative remodeling and inflammation of the pulmonary arterioles that lead to increased pulmonary vascular resistance, right ventricular hypertrophy, and failure. Pulmonary vascular tone is regulated by a balance between vasoconstrictor and vasodilator mediators, and a shift in this balance to vasoconstriction is an important component of PH pathology, Therefore, the mainstay of current pharmacological therapies centers on pulmonary vasodilation methodologies that either enhance vasodilator mechanisms such as the NO-cGMP and prostacyclin-cAMP pathways and/or inhibit vasoconstrictor mechanisms such as the endothelin-1, cytosolic Ca²⁺, and Rho-kinase pathways. However, in addition to the increased vascular tone, many patients have a “fixed” component in their disease that involves altered biology of various cells in the pulmonary vascular wall, excessive pulmonary artery remodeling, and perivascular fibrosis and inflammation. Pulmonary arterial smooth muscle cell (PASMC) phenotypic switch from a contractile to a synthetic and proliferative phenotype is an important factor in pulmonary artery remodeling. Although current vasodilator therapies also have some antiproliferative effects on PASMCs, they are not universally successful in halting PH progression and increasing survival. Mild acidification and other novel approaches that aim to reverse the resident pulmonary vascular pathology and structural remodeling and restore a contractile PASMC phenotype could ameliorate vascular remodeling and enhance the responsiveness of PH to vasodilator therapies.

INTRODUCTION

Pulmonary hypertension (PH) comprises a heterogeneous group of disorders with diverse etiology and pathogenetic mechanisms. PH affects individuals of all ages and is an expanding public health problem in the United States and globally. In healthy subjects with normal pulmonary circulation, the mean pulmonary artery pressure (mPAP) is 14.0 ± 3 mmHg with an upper limit of 20 mmHg. In all forms of PH there is disruption of pulmonary vascular homeostasis and increased mPAP >20 mmHg (1). In severe PH, mPAP could reach ≥35 mmHg. Clinical symptomatology of PH includes dyspnea on exertion that progressively worsens to stages I–IV as described in the World Health Organization (WHO) functional classification (Table 1) (2). PH progresses rapidly and eventually leads to right ventricular hypertrophy and failure that are ultimately fatal (3). Despite promising therapeutic approaches, the mortality rates from PH remain unacceptably high and could be accelerated by COVID-19-related complications. Prognosis of PH is poor and early detection and management is critical, making it important to understand the underlying factors and mechanisms.

Table 1.

WHO functional classification of pulmonary hypertension

Functional Class	Symptomatology
I	No limitations of usual physical activity
II	No symptoms at rest, but dyspnea, fatigue, or chest pains during usual activity
III	No symptoms at rest, but limitations of usual activity due to dyspnea, fatigue, chest pain, or syncope
IV	Symptoms at rest

See Galie et al. (2). WHO, World Health Organization.

Several underlying cardiopulmonary and autoimmune disorders as well as genetic, racial, gender-related, and environmental factors have been suggested as predisposing factors in PH. Also, extensive research has helped to identify some of the pathophysiological components of PH including increased pulmonary arteriolar tone, and pulmonary vascular wall thickening, remodeling, fibrosis, and inflammation (2, 4). Specifically, substantial progress has been made in our understanding of the mechanisms of dysregulated pulmonary vascular tone in both human PH and preclinical animal models. This knowledge was successfully leveraged to develop vasodilator therapies that are variably effective in improving symptoms and prolonging survival among patients with PH. For instance, identifying the alterations in endothelium-derived vasoactive mediators such as nitric oxide (NO), prostacyclin (PGI₂) and endothelin-1 (ET-1) have led to their therapeutic targeting using phosphodiesterase-5 (PDE5) inhibitors (PDE5i), cGMP, and PGI₂ analogs, and ET-1 antagonists (5–9). Combination treatments targeting complementary endothelium-dependent pathways also aim to enhance the vasodilation responsiveness of the PH circulation to levels better than those achieved by each of the vasodilators when used separately (10–12). In addition to endothelial cell dysfunction, enhanced pulmonary arterial smooth muscle cell (PASMC) contraction mechanisms such as Ca²⁺ and Rho-kinase could enhance pulmonary vascular tone in PH. Aberrant transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP) signaling, increased proliferation of PASMCs and fibroblasts, excessive extracellular matrix (ECM) deposition, fibrosis, and inflammation also contribute to abnormal pulmonary vascular remodeling in PH. Therefore, many patients with PH do not respond adequately to vasodilators and their quality of life remains markedly compromised due to progressive dyspnea and exertional fatigue that could lead to ultimate immobilization.

Although PH is increasingly recognized as a complex disease with different underlying mechanisms and multiple pulmonary and cardiovascular organs involved, current therapeutic approaches remain primarily focused on correcting the disrupted pulmonary vascular homeostasis and reducing pulmonary vascular resistance (PVR) by counterbalancing the residual vasoconstriction using vasodilation rather than targeting key regulators of the cellular pathology. Although therapeutic efforts should also target other components of the PH disease including the right ventricle response to high PVR, skeletal muscle dysfunction, and other extrapulmonary manifestations, there remains an unmet clinical need to further examine, evaluate, and enhance the effectiveness of current therapeutic approaches for PH. In this respect, it is increasingly appreciated that a strategy to restore the structural and functional integrity of the pulmonary vessel wall could open new avenues for PH therapies.

This review will discuss reports in PubMed and Medline databases and data from our laboratories with emphasis on the current understanding of the cellular and molecular mechanisms of the disrupted pulmonary vascular homeostasis in PH, the current therapeutic approaches, and implications for potential novel targets. Clinical data in human PH and preclinical data in experimental models of PH will be discussed. The review will highlight the various predisposing factors of PH, the pathological components of the disease including pulmonary vascular dysfunction, remodeling, fibrosis, and inflammation, and the underlying vascular and cellular mechanisms. The review will also describe how the interaction between different cell types and overlapping mechanisms could add to the complexity of PH pathology. Further understanding of these mechanisms should help to identify new targets and develop novel therapeutic strategies to restore pulmonary vascular homeostasis in PH.

PH CLASSIFICATION, GROUPS, AND EPIDEMIOLOGY

The WHO guidelines classify patients with PH into five groups (Table 2) (13, 14). Pulmonary arterial hypertension (PAH, group 1 PH) is uncommon and is estimated to affect between 10 and 50 people per million worldwide. PAH is associated with extensive PASMC proliferation, vascular remodeling, and obliteration of the small pulmonary arterioles, leading to progressive increase in PVR (15).

Table 2.

World Health Organization clinical classification of PH

Group	Mechanisms	Examples
1	Pulmonary arterial hypertension	Idiopathic, heritable, drug-induced, viral (HIV) or parasitic disease (schistosomiasis), connective tissue disorders, liver cirrhosis, congenital heart disease, PPHN
2	PH due to left heart failure	Left-sided atrial, ventricular, or valvular disease
3	PH due to chronic hypoxemic lung disease	COPD, interstitial lung disease, obstructive sleep apnea, high-altitude, developmental lung diseases
4	Chronic thromboembolic PH	Pulmonary emboli
5	Miscellaneous or unclear	Sickle cell disease, sarcoidosis, metabolic disorders, renal failure

See Gelzinis (13) and Walter (14). COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; PPHN, persistent pulmonary hypertension of the newborn.

Although PH is considered a rare disease (fewer than 1 in 2,000 individuals in Europe and fewer than 200,000 in the United States, with 500–1,000 new cases per year), it affects individuals of all ages and is an expanding public health problem in the United States and globally as the population ages (16). It is estimated that 1% of the global population suffers from PH, and this percentage rises to 10% in individuals over the age of 65 yr (16). The prevalence of PH in the United States is estimated at 109/million under the age of 65 and 451/million over the age of 65 (17). Importantly, 80% of the affected global population live in developing countries and their disease is associated with uncorrected congenital heart disease, rheumatic disease, HIV, and parasitic disease (schistosomiasis) (16). The increasing prevalence with advancing age is primarily due to PH in association with left-sided heart disease and lung disease. Among patients with PH in developed countries, the 1-year survival is 83%–91% and the 3-year survival is 58%–72.1% (18, 19). The disease burden in the pediatric population is less well defined with data derived primarily from regional registry cohorts. The estimated prevalence is 20–40 cases per million in Europe (20, 21) and 26–33 per million children in the United States (22). One study in the United States has reported that PH is associated with 0.13% of pediatric hospitalizations and with 5.9% of all-cause mortality (23). In the pediatric population, there is also increasing proportion of patients with PH without associated congenital heart disease especially among surviving preterm infants with or without bronchopulmonary dysplasia (23, 24).

PH PREDISPOSING FACTORS

PH is a complex disorder and often a manifestation of an underlying severe cardiopulmonary, autoimmune, or metabolic disease. There is a diverse group of predisposing genetic and environmental factors that contribute to the various morbidities associated with each group of PH. Bacterial and viral infection, air pollution, and nicotine and E-cigarette smoking are important environmental factors that could predispose to PH.

Specifically, although PAH is uncommon, several predisposing genetic, gender-related, and environmental factors have been suggested. Mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene could be responsible for 80% of heritable PAH and 20% of sporadic PAH. Disruption of BMPR2 signaling is observed in different cells of the pulmonary vasculature including endothelial cells, PASMCs, and fibroblasts. BMPR2 also regulates the immune system, and disruption of BMPR2 signaling disrupts the immune response and increases inflammation (25). Loss of function KCNK3 gene mutation could also alter the circulating immune cells and the inflammation response and enhance susceptibility to PAH (26). Whole exome sequencing (WES) has identified SRY-related HMG box transcription factor (SOX17) as a risk gene and suggested that its deficiency may predispose to PAH (27). Also, WES in two unrelated families with PAH cases showed that TNIP2 and TRAF2 gene loss of function could promote pulmonary vascular remodeling through exaggerated NF-κB signaling (28). WES in 242 Japanese patients with familial or sporadic PAH identified a heterozygous substitution change involving c.226G>A (p.Gly76Ser) in tumor necrosis factor receptor superfamily 13B gene (TNFRSF13B) in six (2.5%) patients. TNFRSF13B controls the differentiation of B cell and secretion of inflammatory cytokines and may be involved in vascular inflammation (29). Also, in a cohort of 79 patients with PAH, four pathogenic/likely pathogenic variants in four different genes (TBX4 encoding T-box 4-containing protein, ABCC8, KCNA5, and GDF2/BMP9) were identified in nine patients (30). Mutant CPS-I A/C rs4399666 minor variant particularly the homozygous CC genotype is frequently distributed among neonates with persistent pulmonary hypertension of the newborn (PPHN) (31). In a cohort of 60 patients with PAH, single-nucleotide polymorphisms (SNPs) for genes encoding the mitochondrial proteins Sirtuin3 (Sirt3) and uncoupling protein 2 (Ucp2) were associated with disease severity and death or need for lung transplant and 10-year outcomes. Also, double knockout mice for Sirt3 and Ucp2 genes show greater severity of PAH, mPAP, right ventricular hypertrophy/dilatation, vascular remodeling, and inflammatory plexogenic lesions compared with wild-type mice (32). Rare variant analysis of 4,241 PAH cases from an international consortium identified seven genes with rare deleterious variants associated with idiopathic PAH including BMPR2, GDF2, TBX4, SOX17, and KDR and two new candidate genes encoding fibulin 2 (FBLN2) and platelet-derived growth factor D (PDGFD) (33).

Epigenetic mechanisms including DNA methylation and histone posttranslational modifications regulate chromatin structure and lineage-specific gene expression during cardiovascular development and disease. Analysis of cells and tissues isolated from patients with PAH and preclinical animal models of PH showed alterations in the levels of multiple histone deacetylases, SIRT1, SIRT3, and bromodomain-containing protein 4 (BRD4) and their strong association to proliferative, fibrotic, and inflammatory phenotypes that lead to pulmonary artery remodeling (34). Also, histone deacetylase 1 is upregulated in monocrotaline (MCT) model of PH, and its inhibition reduces ECM accumulation, pulmonary arterial remodeling, right ventricular systolic pressure (RVSP), and right ventricle hypertrophy index. The underlying mechanism involves upregulation of miR-34a and modulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS), specifically MMP-9/TIMP-1 and MMP-2/TIMP-2 activity (35).

Regarding gender differences, there is a predominance of young females affected by heritable PAH, but among patients with PAH, women show better prognosis and right ventricular function and higher survival rates than men (36, 37). The increased frequency of PAH among women has been partly related to increased expression/activity of the long-noncoding RNA X-inactive-specific transcript (Xist), which is essential for X-chromosome inactivation and dosage compensation of X-linked genes, leading to sexual dimorphism in female pulmonary artery endothelial cells (38). The increased susceptibility to PAH in women has also been related to estrogen/estrogen receptor signaling. Paradoxically, estrogen may have protective effects in animal models of PH, as the pathology is exaggerated in ovariectomized female animals. In isolated human pulmonary artery and PASMCs from non-PH subjects and group 3 patients with PH, estrogen pretreatment has shown variable effects ranging from no effect to enhancement of PGI₂ synthesis and the levels of 6-keto-prostaglandin F1α (PGI₂ stable metabolite), the PGI₂ IP-receptor, and arachidonic acid-induced relaxation (39). Also, 17β-estradiol acting on estrogen receptor-α attenuates right ventricular failure in MCT rat model of PH by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent pathway (40). In chronic hypoxia (Hx) rat model of PH, treatment with the estrogen metabolite 2-metyhoxyestradiol reduced RVSP and pulmonary vascular remodeling (41). This “estrogen paradox” may be related to differences among species in the synthesis/metabolism of estrogens, the types, distribution and function of estrogen receptor, the post-estrogen receptor genomic and nongenomic signaling pathways, and changes in estrogen signaling during the development and progression of PH (36). These conflicting observations make it important to further examine the sex differences and the potential protective effects of estrogen in PH.

In addition to genetic and gender-related factors, a large number of drugs and toxins can induce PAH in susceptible individuals by known and unknown mechanisms. The first drugs observed to be causally related to PAH were the amphetamine-like appetite suppressants aminorex, fenfluramine, and dexfenfluramine. In addition to the use of anorexigens, there is increasing recognition of PAH susceptibility in association with the use of illicit drugs such as cocaine and amphetamines as well as medically prescribed drugs such as diazoxide and fluoxetine (42). Amphetamine-like appetite suppressants such as fenfluramine and dexfenfluramine could contribute as predisposing factors to PH pathogenesis through displacing norepinephrine and neurotransmitters. Other proposed underlying mechanisms include aberrant serotonin release, K⁺ channel dysregulation, and genetic predisposition.

PATHOPHYSIOLOGICAL COMPONENTS OF PH

PAH involves residual pulmonary vasoconstriction, progressive small vessel remodeling and obliteration, large vessel thickening and obstruction, and development of plexiform lesions (43). Increased PVR in PH is the result of 1) increased pulmonary arteriolar tone due to vasoconstriction and 2) structural remodeling of the pulmonary arterioles that includes PASMC proliferation perivascular fibrosis and inflammation causing obliteration of the vessel lumen and fixed obstruction (Fig. 1). Increased release of neurovascular mediators from pulmonary nerve endings could contribute to increased pulmonary vasoconstriction. Endothelial cell dysfunction is also a major factor in PAH (44). An imbalance between endothelial-derived vasodilators and vasoconstrictors has long been recognized as a modifiable contributor to the disease process, and consequently has been the target of most of the current vasodilator therapies (45). Changes in PASMC contraction pathways such as cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Rho-kinase have also been linked to PH, and their targeting is promising, but less well developed (46, 47). The structural remodeling of pulmonary arterioles involves a complex interplay of multiple resident and circulating cells as well as humoral factors that collectively disrupt all vascular layers of the pulmonary arterioles and render them less responsive to current vasodilator therapies (48). Changes in growth factors, inflammatory elements, and the tissue pH could promote PASMC phenotypic switching from contractile to synthetic phenotype and activate multiple mediators of structural pulmonary vascular remodeling in PH (49, 50). This is an area of enormous potential as better mechanistic insights are accumulating.

Figure 1.

Vascular mechanisms of pulmonary hypertension (PH). Genetic factors or underlying left cardiac, pulmonary hypoxic, thromboembolic, or autoimmune disease led to hypoxia and targeting of various cells in the pulmonary vasculature. In endothelial cells, decreased release of endothelium-derived relaxing factors (EDRFs) reduces pulmonary arterial smooth muscle cell (PASMC) relaxation, whereas increased endothelium-derived constricting factors (EDCFs) increases PASMC contraction, leading to increased pulmonary vascular tone. Increased sympathetic nerve ending activity could also increase pulmonary vasoconstriction. Phenotypic switching of PASMCs from contractile to synthetic phenotype promotes PASMC growth, proliferation, migration, and resistance to apoptosis, and leads to increased pulmonary arteriole medial thickening. Activation of fibroblasts and pericytes causes increases in the release of transforming growth factor-β (TGFβ) and matrix metalloproteinases (MMPs), decreases in tissue inhibitors of metalloproteinases (TIMPs), and increases in extracellular matrix (ECM), collagen deposition, fibrosis, and pulmonary arteriolar remodeling. Inflammatory cell infiltration, increased release of inflammatory cytokines, and macrophage polarization lead to pulmonary arteriole inflammation and further remodeling. The increased pulmonary arteriolar tone, medial thickening, and remodeling lead to increased pulmonary vascular resistance (PVR), pulmonary hypertension (PH), and right ventricular failure. Current vasodilator therapies aim at counteracting the residual pulmonary arteriolar narrowing, whereas mild acidification, antifibrotics, and anti-inflammatory drugs could provide adjuvant therapies to minimize pulmonary arteriolar obliteration and reduce PVR and PH. ECM, extracellular matrix; EDHF, endothelium-derived hyperpolarizing factor; ET-1, endothelin-1; ET_AR, endothelin receptor type A; HIF, hypoxia-inducible factor; NO, nitric oxide; PDE5, phosphodiesterase-5 inhibitor; PGI₂, prostacyclin.

Rodent models of experimental PH recapitulate to a certain extent the histologic features of human PH and provide a useful tool to study the efficacy of novel therapeutic approaches (51–55). The MCT and Hx rat models are particularly useful in the evaluation of antiproliferative and proapoptotic therapies since medial hypertrophy of the pulmonary arterioles is a key feature in these PH models (56–59). Of note, although chronic exposure to Hx is widely used as an animal model of PH, Hx may induce different genes in the lungs of rats versus mice (60). The sugen 5416/hypoxia (Su5416/Hx) rat model of PH more closely recapitulates the human pathology due to its progressive nature, formation of plexiform lesions, and ultimate lethality (61–64). Sugen 5416 (semaxanib) is an antineoplastic drug with tyrosine kinase inhibitor and vascular endothelial growth factor receptor (VEGFR)-2 inhibitor properties (65). Su5416/Hx in rats and mice sets off a cascade of events that lead to structural remodeling of the pulmonary arterioles and progressively worsening hemodynamics and plexiform lesions that are not seen in any other model of PH (66, 67). As such, this model has been very useful to study the pathogenetic mechanisms of PH and identify novel therapeutic targets.

INCREASED SYMPATHETIC TONE AND NEUROVASCULAR MEDIATORS IN PH

Neurohormonal imbalance is observed in many cardiovascular disorders and has been associated with worse prognosis and survival in patients with PH. Increased neurovascular release of noradrenaline is expected to increase pulmonary vasoconstriction. However, we have shown that phenylephrine was less potent in producing vasoconstriction in pulmonary arteries of Su5416/Hx versus control rats (68), suggesting decreased sensitivity of the α-adrenergic receptors. This could be partly explained by chronic increase in sympathetic discharge from the nerve endings leading to desensitization of the α-adrenergic receptors, making it important to further examine pulmonary sympathetic activity in PH.

Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are released from sensory C fiber nerve endings in pulmonary microvessels likely through activation of transient receptor potential vanilloid type-1 channel (TRPV1) (69). CGRP is a vasodilator (via cAMP) and substance P is a vasoconstrictor or vasodilator depending on which cell it acts on, but it also promotes vascular smooth muscle cell (VSMC) proliferation.

Dysregulation of the renin-angiotensin-aldosterone system (RAS) has also been implicated as a causative factor in PH. Angiotensin II (ANG II) can exert deleterious effects on the pulmonary vasculature by promoting vasoconstriction, VSMC proliferation, and vascular inflammation (70). Therapies targeting RAS, such as angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor type 1 (AT₁R) blockers, may be useful in PH, but the likelihood of these RAS modulators causing systemic hypotension is a serious consideration. Other studies have shown that activation of angiotensin converting enzyme 2 (ACE2), which converts ANG II to Ang-(1–7), can also induce beneficial outcomes in PH through vasodilation and inhibition of inflammation (71–74).

Transcatheter pulmonary artery denervation by applying circumferential radiofrequency ameliorated mPAP and PVR in some patients with left heart failure with reduced ejection fraction and PH (75). Although pulmonary artery denervation could reduce the sympathetic activity in the pulmonary circulation, the sympathetic system is crucial to facilitate bronchodilation and oxygen uptake, making it important to further evaluate the benefits of this approach in large-scale multicenter randomized controlled clinical trials. In effect, some studies suggest potential benefits of parasympathetic stimulation in PH, and show that oral cholinesterase inhibitor donepezil (Aricept), a drug used to treat Alzheimer’s disease, enhances parasympathetic activity, and reduces mPAP, RVSP, and pulmonary arterial remodeling in MCT-treated rat model (76). Although this study did not address potential detrimental effects of parasympathetic activation such as bronchoconstriction, these effects will need to be considered in the evaluation of translatability to clinical application.

ENDOTHELIAL DYSFUNCTION AND ENDOTHELIUM-DERIVED VASOACTIVE MEDIATORS IN PH

PH is associated with dysfunction of different cells of the pulmonary vascular wall, including endothelial cells. Maintenance of pulmonary vascular homeostasis involves complex endocrine, autocrine, and paracrine interactions in the vascular milieu, and the endothelium is a major determinant of vascular homeostasis. The intact endothelium has thrombo-resistant and anti-inflammatory properties, and its barrier function prevents extravasation of fluid and macromolecules and penetration of inflammatory cells such as neutrophils and monocytes into the vessel wall. In response to a variety of injurious stimuli such as Hx, shear stress, or inflammatory cytokines, the endothelium assumes an activated state, loses its barrier function and thrombo-resistant properties, and releases growth factors and adhesion molecules that contribute to vascular wall remodeling, in situ thrombosis, and PH pathology. Endothelial cell dysfunction has been described in both clinical PH and preclinical animal models especially in the context of dysfunctional BMPR2 signaling (77) with resultant disruption of PPARγ and p53 transcriptional regulation of downstream genes involved in endothelial cell survival and angiogenesis (78–80). Also, endothelial cell deficiency in the iron-sulfur biogenesis protein frataxin could decrease iron-sulfur clusters and lead to endothelial cell senescence and PH (81). The endothelium is known to release endothelium-derived relaxing factors such as nitric oxide (NO), prostacyclin (PGI₂), and endothelium-derived hyperpolarizing factor as well as vasoconstricting factors such as endothelin-1 (ET-1) and thromboxane A₂ (TXA₂). Endothelial cell dysfunction could lead to imbalance between vasodilator and vasoconstrictor factors and increased pulmonary vasoconstriction and PVR.

Nitric Oxide

NO is a major effector of endothelium-dependent vasodilation (Fig. 2). NO is produced by the enzymatic action of endothelial NO synthase (eNOS or NOS2) on l-arginine and diffuses to the adjacent VSMCs where it activates soluble guanylate cyclase that generates cyclic GMP (cGMP). cGMP activates protein kinase G that induces VSMC relaxation by promoting Ca²⁺ extrusion and reducing Ca²⁺ sensitivity of the contractile proteins. Phosphodiesterase-5 (PDE5) breaks down and terminates the vasodilator effects of cGMP. Downregulation of the NO-cGMP relaxation pathway is an important factor in the increased pulmonary vasoconstriction in PH. Expression of endothelial NOS is reduced in patients with PAH (82). Acetylcholine (ACh)-induced relaxation is reduced in isolated lung preparation of Hx rats (83). Also, ACh-induced relaxation and the NO-cGMP pathway are reduced in pulmonary arteries of the MCT, Hx, and Su5416/Hx rat models (68, 84). ACh-induced NO production is reduced in the Su5416/Hx rat model (68), likely due to decreased expression/activity of NOS. The reduced ACh-induced relaxation could involve decreased NO bioavailability due to increased reactive oxygen species during chronic Hx-induced vascular remodeling and PH (85). The reduced ACh relaxation in PH models could also be due to structural changes in the pulmonary vascular wall that decrease the responsiveness of PASMCs to vasodilators. This is supported by our observation that pulmonary artery relaxation to the exogenous NO donor sodium nitroprusside is reduced in the MCT, Hx, and Su5416/Hx rats (68, 84). This is also consistent with reports that both endothelium-dependent and -independent relaxation are reduced in the MCT-induced rat model of progressive lung injury (86). Some studies found that endothelium-independent vasodilation by sodium nitroprusside was fully active in the Hx rat model (83), and the different findings could be related to the vascular preparation used (pulmonary artery vs. isolated perfused lung) or other factors affecting the responsiveness to the exogenous NO donors, including changes in PDE5 or protein kinase G activity.

Figure 2.

Nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pulmonary arterial relaxation pathway. l-Arginine is taken up by pulmonary arterial endothelial cells to be converted by endothelial NO synthase (eNOS) to NO and l-citrulline. NO diffuses into pulmonary arterial smooth muscle cells (PASMCs), where it activates soluble guanylate cyclase that converts GTP into cGMP. cGMP activates cGMP-dependent protein kinase or protein kinase G (PKG) leading to decreased [Ca²⁺]_cyt, inhibition of myosin light chain kinase (MLCK) activity, and PASMC relaxation. The NO-cGMP pathway could be enhanced by l-arginine supplementation, NOS induction by gene therapy, NO inhalation, NO donors, or guanylate cyclase activators. The NO-cGMP pathway could also be augmented by inhibiting phosphodiesterase-5 (PDE5) and preventing the conversion of cGMP to GMP, or by inhibiting arginase and preventing the conversion of l-arginine to ornithine and urea. cGMP, cyclic guanosine monophosphate; PDE5i, PDE5 inhibitor.

Considering the important role of NO-cGMP in pulmonary vascular relaxation, many of the current therapies involve modulation of this pathway (Table 3). NO inhalation ameliorates PH and improves organ functions in children undergoing corrective surgeries for congenital heart disease and in adults after heart valve surgeries (87). The PDE5i sildenafil may facilitate weaning from inhaled NO in children with PAH following surgery for congenital heart disease (7). Combined use of PDE5i and nitrates has also been considered in ameliorating PAH in patients with heart failure (8). A meta-analysis of clinical trials has shown that sildenafil administration by nasogastric feeding can improve PPHN (88). Sildenafil treatment also improves MCT-induced PH in rats (53). Enhancing the NO-cGMP pathway using l-arginine supplementation, the NO donor molsidomine or PDE5i sildenafil also ameliorated right ventricle hypertrophy and myocardial fibrosis and apoptosis in Hx rat model of PH (89). Antioxidants and modulators of mitochondrial oxidative phosphorylation pathways reduce reactive oxygen species and could increase NO bioavailability and promote vasodilation. In support, dichloroacetate (DCA), an inhibitor of the mitochondrial pyruvate dehydrogenase kinase, the gatekeeping enzyme of glucose oxidation, has been shown to improve hemodynamics and functional capacity in a subset of patients with PAH. Of note, other patients who had inactivating mutations of two genes encoding mitochondrial enzymes (SIRT3 and UCP2) were not responsive to DCA, suggesting a relationship between mitochondrial pyruvate dehydrogenase kinase activity and genetic susceptibility to PAH (90).

Table 3.

Endothelial-derived vasoactive mediators, signaling pathways, and related medications for PH

Mediator	Receptor/Target in Smooth Muscle	Postreceptor Pathways	Related Approved Medications
Vasodilators
NO	Guanylate cyclase	cGMP, PKG -PMCA, ↑Ca2+ extrusion -Phosphorylation of MLCK, ↓MLCK activity, ↓ Ca2+ sensitivity	-Inhaled NO in acute PH -Guanylate cyclase stimulators, Riociguat -PDE5i, Sildenafil, Tadalafil, Vardenafil
PGI2	IP receptor	cAMP, PKA -PMCA, ↑Ca2+ extrusion -Phosphorylation of MLCK, ↓MLCK activity, ↓Ca2+ sensitivity	IP receptor agonists, Epoprostenol, Treprostinil, Beraprost, Iloprost, Selexipag
Vasoconstrictors
ET-1	ETAR	-Ca2+ release from SR -Ca2+ influx via channels -Ca2+/CAM, activates MLCK -Rho-A/ROCK pathway, PKC pathway, inhibit MLC phosphatase	-Nonselective ETR antagonists, Bosentan, Macitentan -Selective ETAR antagonists. Ambrisentan
TXA2	TP receptor	-Ca2+ release from SR -Ca2+ influx via channels -Ca2+/CAM, activates MLCK -Rho-A/ROCK pathway, PKC pathway, inhibit MLC phosphatase	TXA2 antagonists Need further evaluation clinically -Daltroban, Domitroban, Sulotroban, Terutroban -Vapiprost, Seratrodast

CAM, calmodulin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ET-1, endothelin-1; ET_AR, endothelin receptor type A; IP, prostacyclin receptor; MLC, myosin light chain; MLCK, MLC kinase; NO, nitric oxide; PDE5, phosphodiesterase-5; PH, pulmonary hypertension; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PMCA, plasma membrane Ca²⁺-ATPase, ROCK, Rho-associated protein kinase; SR, sarcoplasmic reticulum; TXA₂, thromboxane A₂.

Other approaches that could enhance the NO-cGMP pathway have been studied. Oral 1-nitro-2-phenylethane and trans-4-methoxy-β-nitrostyrene are novel soluble guanylate cyclase stimulators that improve mPAP, pulmonary hemodynamics, and pulmonary vascular function and remodeling, and reduce RVSP and right ventricular hypertrophy in MCT rat model (91, 92). We have shown that ACh relaxation and NO production are improved in pulmonary arteries of Su5416/Hx rats by inducing mild acidification using the carbonic anhydrase inhibitor acetazolamide (ACTZ) (93). The ACTZ beneficial effects could be related to improved NOS activity, increased NO bioavailability (94), or improved PASMC responsiveness to NO and other vasodilators (93).

Prostacyclin

Prostacyclin (PGI₂) is a member of the prostanoid family of eicosanoids derived from arachidonic acid by the sequential actions of phospholipase A2, cyclooxygenase (COX), and prostaglandin (PG) synthases in endothelial cells. Endothelial-derived PGI₂ binds to the prostacyclin receptor (IP) in VSMCs (95) and stimulates a G protein-coupled increase in cAMP that activates protein kinase A, resulting in decreased [Ca²⁺]_cyt and enhanced vasorelaxation. PGI₂ may also reduce VSMC contraction and PVR by inhibiting Rho-kinase. PGI₂ through intracrine signaling may also target nuclear peroxisome proliferator-activated receptor (PPAR) and in turn regulate gene transcription, VSMC proliferation, and pulmonary vascular remodeling (96, 97).

Studies in patients with PH and preclinical animal models have identified deficiencies in different components of the PGI₂ pathway, and demonstrated therapeutic benefits of enhancing PGI₂ signaling, and consequently led to clinical use of PGI₂ analogs in PH (97–104). Patients with severe PAH have reduced IP receptor expression in their remodeled PASMCs (97). Studies in Hx mouse model have shown that IP receptor KO mice develop more severe PH and vascular remodeling compared with WT mice, and suggested that modulation of PGI₂ synthesis and IP receptor activity could reduce pulmonary vascular remodeling and PH (97). In support, overexpression of pulmonary PGI₂ synthase in transgenic mice protects against the development of Hx-induced PH (99).

Different parenteral, inhalation (epoprostenol), and oral prostanoids are currently available as PH therapies. Oral IP receptor agonist selexipag reduces disease progression and morbidity/mortality in patients with PAH (105). Treprostinil palmitil is a long-acting inhaled pulmonary vasodilator prodrug of treprostinil. Early initiation and higher doses of intraperitoneal (IV) or subcutaneous (SC) prostanoids and combination with other PAH-targeted therapy showed favorable outcome, and transition from parenteral to oral or inhaled prostanoid therapies was safe on the long-term in children with PAH (106). Inhaled iloprost may also be better than inhaled NO in offering an effective, safe, and pulmonary-selective vasodilation strategy for the treatment of group 2 patients with PH and left heart disease (9).

Endothelium-Dependent Hyperpolarization

Endothelium-dependent hyperpolarization involves activation of K⁺ channels in vascular cells (101, 107). In pulmonary artery endothelial cells, K⁺ channel activity controls resting membrane potential, Ca²⁺ entry, and the release of different vasoactive factors. Transient receptor potential vanilloid-4 (TRPV4) channels activity in endothelial cell caveolae plays an important role in maintaining normal mPAP. Dysregulation of K⁺ channel expression/activity by Hx or other vasoactive factors could contribute to the pathogenesis of PH. Pulmonary arteries from patients with PH and mouse models show upregulation of iNOS and NOX1 enzymes and increased peroxynitrite in endothelial cell caveolae that could affect caveolin-1 and reduce TRPV4 channel activity in PH (108).

Endothelium-dependent hyperpolarization spreads through myoendothelial junctions to underlying VSMCs. In PASMCs, K⁺ channels determine membrane potential, with increased activity leading to membrane hyperpolarization and pulmonary vasodilation whereas decreased activity leads to membrane depolarization, Ca²⁺ entry, and pulmonary vasoconstriction. K⁺ channel activity could also affect PASMC phenotype, proliferation, and survival/apoptosis.

Enhancing K⁺ channel expression/activity could be an attractive strategy to reduce PVR in patients with PH (109). Many of the existing vasodilator treatments indirectly increase K⁺ channel activity (110–112), and direct modulators of channel K⁺ channel expression/activity have also been proposed as potential therapeutic approaches in PH. For instance, genetic approaches such as gene therapy or administration of antago-miRs have been considered to increase K⁺ channel expression in PH (113, 114). Selective and nonselective K⁺ channel activators have also shown beneficial effects ex vivo (115, 116) and in preclinical models (117, 118). In addition, nutritional factors, antioxidants, and other compounds have shown K⁺ channel activating effects that could be applied therapeutically (119–122). Further understanding of the diversity of K⁺ channel types and the complexity of mechanisms by which their dysregulation contributes to PH pathogenesis as well as consideration of potential off-target effects of these K⁺ channel modulators are needed before clinical translation.

Hydrogen Sulfide

Hydrogen sulfide (H₂S) is a major transmitter with regulatory effects in the cardiovascular system and the pulmonary circulation. H₂S is involved in pulmonary vascular cell homeostasis and could counterbalance the Hx response and the disruption in mitochondrial bioenergetics induced during PH development. For instance, H₂S modulates ATP-sensitive K⁺ channel (K_ATP) activity and in turn promotes pulmonary artery relaxation. H₂S also reduces oxidative stress, the inflammatory response, and pulmonary artery remodeling. Alteration in H₂S biogenesis could contribute to the pathogenesis of PH. H₂S may promote protective effects against PH, and H₂S-releasing agents could be a new therapeutic strategy in PH (123).

Endothelin-1

ET-1 is a potent endothelium-derived vasoconstrictor and mitogen for VSMCs. ET-1 binds to G protein-coupled endothelin receptor type A and type B on the adjacent VSMCs causing increases in [Ca²⁺]_cyt, myosin light chain (MLC) phosphorylation, and vasoconstriction. ET-1 also activates Rho-Kinase and protein kinase C leading to inhibition of MLC phosphatase, increased myofilament sensitivity to Ca²⁺ and further vasoconstriction (124–126).

Studies have shown elevated ET-1 levels in patients with PAH (127) and animal models of PH (128) and suggested a causal relationship with disease pathogenesis, and this led to the development of endothelin receptor antagonists as therapeutic agents for PAH (129–132). Bosentan, macitentan, and aprocitentan are dual ET-1 receptor antagonists to both endothelin receptor type A (ET_AR) and endothelin receptor type B (ET_BR). Importantly, ET_BRs are also expressed in endothelial cells and mediate endothelin clearance and NO and PGI₂ release. ET_BR-deficient mice show exaggerated response to Hx and PH (133). To avoid interference with the beneficial vasodilator endothelial ET_BRs, selective ET_AR blockers have been developed. ET_AR blockade partially reverses neonatal hypoxic pulmonary vascular remodeling (134) and reduces contraction in pulmonary arteries from Hx rats (128). However, clinically, selective ET_AR antagonist has not been proven to be superior to nonselective ET antagonists in ameliorating PAH (135).

Analysis of blood samples from the pulmonary artery and systemic vessels of children with PAH showed a wide range of plasma levels of ET-1 and big ET-1, suggesting possible changes in the net release or lung clearance of ET-1. Also, in neonates with PPHN, we found increased levels of circulating ET-1 that decreased in response to treatment with inhaled NO (136). However, in other studies, ET-1 and big ET-1 levels showed no correlation with the clinical and hemodynamic parameters or the severity of PAH and were not able to predict outcome (137), supporting the involvement of other factors in the disease process.

Thromboxane A₂

Increased TXA₂ activity may play a role in the pathogenesis of cardiopulmonary disorders including atherosclerosis, myocardial infarction, stroke, bronchial asthma, and PH. Studies have empirically determined a dose of the TXA₂ analog U46619 that could induce PH in pigs without causing systemic hemodynamic disturbances, making it an attractive model to evaluate the efficiency of different therapeutic approaches of PH in large animals (138). Several TXA₂ antagonists have been developed, but their effectiveness and safety need to be further evaluated clinically in PH.

Heme Oxygenase-1

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme responsible for degradation of heme to biliverdin, free iron, and carbon monoxide. VSMCs through increased HO-1 activity could release carbon monoxide that exerts paracrine effects on the adjacent endothelial cells. Carbon monoxide is a potent vasodilator. HO-1 via its products inhibits oxidative stress and inflammation (139). HO-1 deficiency results in endothelial damage with elevation of thrombomodulin and von Willebrand factor (140).

Stimuli that cause oxidative stress such as peroxynitrite and Hx modulate HO-1 expression (141). HO-1 enzymatic products have shown beneficial cardiopulmonary actions in chronic Hx (142). Also, enhancement of endogenous HO-1 activity improved the hemodynamics and mPAP in Hx rat model (143).

IMPAIRED VSMC CONTRACTION PATHWAYS IN PH

Changes in VSMC contraction mechanisms could also contribute to increased pulmonary vascular tone and PVR. Studies have shown enhanced ET-1-induced pulmonary vasoconstriction in the Fawn-Hooded rat model of spontaneous PAH (144). Also, pulmonary vessels are hyperresponsive in perfused lungs from MCT-treated rats (145), and pulmonary vasomotor tone is increased in Hx rat model (146, 147). Chronic Hx is thought to increase the production/activity of ET-1 and ANG II in the lung leading to increased vasoconstriction (146). On the contrary, we have found that the contraction of pulmonary artery, but not the aorta and systemic mesenteric vessels, to phenylephrine and high KCl depolarizing solution is reduced in the MCT, Hx, and Su5416/Hx rat models (68, 84), indicating specific changes in the pulmonary circulation. The reduced pulmonary artery contraction in MCT and Hx rats may not be due to decreased sensitivity/number of α-adrenergic receptors because phenylephrine was equally potent in control, MCT, and Hx rats, and the contraction to high KCl, a receptor-independent response, was also reduced. Other studies have shown that ET-1-induced pulmonary artery contraction is also reduced in the Hx rat model (128), suggesting a reduction in a common postreceptor contraction pathway or a switch in PASMCs from a contractile to synthetic phenotype. The differences in the pulmonary vascular contractile response in different studies could be due to differences in the animal model, the vasoconstrictor studied (phenylephrine and KCl vs. ET-1 and ANG II) or the vascular preparation used (pulmonary artery versus isolated perfused lung), making it important to further examine the molecular mechanisms of VSMC contraction and their potential changes in PH.

Ca²⁺-Dependent Contraction Pathways in PH

Ca²⁺ is a major determinant of VSMC contraction (Fig. 3). In PASMCs, vasoconstrictor agonists such as ET-1 and TXA₂ activate G protein-coupled receptor (GPCR) leading to activation of plasma membrane phospholipase C and increased generation of inositol trisphosphate (IP₃). IP₃ releases Ca²⁺ from the sarcoplasmic reticulum (SR). Vasoconstrictor agonists also activate Ca²⁺ influx though plasma membrane Ca²⁺ channels. Increased [Ca²⁺]_cyt binds calmodulin and activates MLC kinase (MLCK) leading to increased MLC phosphorylation, actin-myosin interaction, and VSMC contraction. Hx could alter the synthesis of or responsiveness to vasoactive factors and in turn causing changes in PASMC electrophysiological properties and signaling pathways (148). Hx could also affect PASMC function by modulating the receptor population, ion channel activity, K⁺ current, and membrane depolarization leading to elevation of [Ca²⁺]_cyt and increased vascular contraction, PVR, and mPAP (146). Ca²⁺-dependent activation of PI₃K/AKT/mTOR pathway could also promote gene expression, PASMC proliferation, and pulmonary artery remodeling (149, 150).

Figure 3.

Interaction between pulmonary arterial smooth muscle cell (PASMC) contraction and relaxation pathways. In PASMCs, vasoconstrictors such as endothelin-1 (ET-1) and thromboxane A₂ (TXA₂) activate their corresponding surface membrane G protein-coupled receptor (GPCR) and stimulate the GTP-binding protein G_q-11 leading to activation of phospholipase C (PLC) and increased generation of inositol-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ causes Ca²⁺ release from the sarcoplasmic reticulum (SR), whereas activation of surface membrane Ca²⁺ channels leads to Ca²⁺ influx and increased [Ca²⁺]_cyt. Ca²⁺ binds calmodulin to activate myosin light chain kinase (MLCK), leading to MLC phosphorylation, increased myosin ATPase activity, actin-myosin interaction, and Ca²⁺-dependent contraction. DAG with or without Ca²⁺ activates different protein kinase C (PKC) isoforms. Activation of GPCRs could also stimulate the GTP-binding protein G_12-13 and guanine nucleotide exchange factor (GEF) leading to stimulation of Rho-A that activates Rho-kinase (ROCK). PKC and ROCK directly or indirectly through phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17) leading to inhibition of MLC phosphatase, thus augmenting MLC phosphorylation and Ca²⁺ sensitization of the contractile proteins. Prostacyclin (PGI₂) is a pulmonary vasodilator acting through prostacyclin receptor (IP) to stimulate adenylate cyclase (AC) and convert ATP to cAMP, which activates protein kinase A (PKA). Also, nitric oxide (NO) diffuses into PASMCs to activate guanylate cyclase (GC) and convert GTP to cGMP, which activates protein kinase G (PKG). Both PKA and PKG stimulate Ca²⁺ extrusion and decrease [Ca₂₊]_cyt, leading to activation of MLC phosphatase, MLC dephosphorylation, and PASMC relaxation. PKA and PKG could also phosphorylate MLCK and decrease its activity, further enhancing PASMC relaxation. PKA may also inhibit ROCK and prevent its Ca²⁺ sensitization mechanisms. Interrupted lines indicate inhibition.

Transient receptor potential channels (TRPCs) are stretch-activated nonselective Ca²⁺-permeable cation channels. TRPC6 channels are upregulated in PASMCs from patients with PAH. Also, 2-aminoethyl diphenyl borniate (2-APB), a nonselective cation channel blocker, and BI-749237, a selective TRPC6 blocker, inhibited acute Hx-induced pulmonary vasoconstriction, and intraperitoneal injection of 2-APB ameliorated PH in animal model, suggesting TRPC6 channel modulation as a novel therapeutic target in PH (151).

Piezo channel superfamily is also a mechanosensitive cation channel responsible for stretch-mediated Ca²⁺ and Na⁺ influx in multiple cell types. Upregulation of Piezo1 has been associated with increased [Ca²⁺]_cyt in PASMCs from patients with idiopathic PAH (152). Interestingly, Piezo1 is upregulated in pulmonary arterial endothelial cells from patients with idiopathic PAH, and Piezo1 mRNA expression and protein levels are increased in the pulmonary artery and PASMCs from animal models of severe PH, supporting a role of mechanosensitive stretch-activated channels and Ca²⁺ influx in the increased vasoconstriction and pulmonary vascular remodeling in PH (153).

Hx may also promote polymerization and activation of the extracellular Ca²⁺-sensing receptor, and a peptide blocking the receptor polymerization was found to reduce its Hx-induced activation and downstream pathways leading to PH (154).

Rho-Kinase Pathway in PH

Vasoconstrictor agonists could activate the RhoA/Rho-associated protein kinase (ROCK) pathway and increase the sensitivity of the contractile apparatus to [Ca²⁺]_cyt (155). ROCK2 levels are greater in PASMCs from patients with idiopathic PAH than normal PASMCs (156). Enhanced ROCK activity and Ca²⁺ sensitization of the contractile proteins has also been demonstrated in preclinical PH models. In Fawn-Hooded rat model of spontaneous PAH, ET-1 promotes vasoconstriction partly by increasing ROCK activity (144). Hx could increase pulmonary vasoconstriction by enhancing ROCK activity in PASMCs. Both ET-1 and serotonin could be involved in enhancing RhoA/ROCK signaling in the pulmonary circulation of Hx rat model (157). Studies have also suggested an important role of ROCK-mediated vasoconstriction in rat models of severe occlusive PAH (147). MCT-treated PH rats also show increased ROCK2 expression in PASMCs (156).

ROCK inhibitors such as fasudil may have the potential to reduce vasoconstriction and vascular remodeling in PH (Table 4). ROCK2 inhibition using specific ROCK2 inhibitor KD025 or its knockdown using siRNA reduced the exaggerated proliferation of PASMCs from patients with PAH. KD025 also attenuated the elevated RVSP in MCT rats, supporting a role of ROCK2 in the pathogenesis of PAH (156). Based on the therapeutic potential of fasudil (ROCK inhibition) and dichloroacetate (inhibition of mitochondrial pyruvate dehydrogenase kinase and oxidative stress) on PAH, studies have explored the effects of fasudil dichloroacetate and found that it attenuated Su5416/Hx-induced PAH, and reduced RVSP, pulmonary artery wall thickness, pulmonary vessel muscularization, perivascular fibrosis, and right ventricular hypertrophy and fibrosis. In PASMCs, fasudil dichloroacetate inhibited Hx-induced proliferation, migration, and contraction to a greater extent than fasudil or dichloroacetate alone, and reduced [Ca²⁺]_cyt, Ca²⁺/calmodulin-dependent MLC phosphorylation and Rho-kinase activity, and restored mitochondrial function (158).

Table 4.

VSMC contraction mechanisms and potential therapies for PH

Mechanism	Sources/Stimulators	Target	Potential Therapies for PH
[Ca2+]cyt	Ca2+ release from SR Ca2+ entry through channels	Ca2+/calmodulin, MLCK, MLC phosphorylation	Ca2+ channel blockers, Nifedipine
ROCK	Rho-A	Phosphorylates CPI-17, inhibits MLC phosphatase, ↑ Ca2+ sensitivity	ROCK inhibitors, Fasudil ROCK2 inhibitors, KD025
PKC	Diacylglycerol, Ca2+	Phosphorylates CPI-17, inhibits MLC phosphatase, ↑ Ca2+ sensitivity	PKC inhibitors, GF 109203X (Bisindolylmaleimide)

CPI-17, protein kinase C-potentiated phosphatase inhibitor protein-17; MLC, myosin light chain; MLCK, MLC kinase; PH, pulmonary hypertension; PKC, protein kinase C; ROCK, Rho-associated protein kinase; SR, sarcoplasmic reticulum.

Protein Kinase C Signaling in PH

Vasoconstrictor agonists also increase diacylglycerol production and activate protein kinase C (PKC), which in turn enhances the Ca²⁺ sensitivity of the contractile proteins (159). PKC activation may be involved in the increased vasoconstriction and vascular remodeling in PH. In Fawn-Hooded rat model of spontaneous PAH, ET-1-induced vasoconstriction could partly involve PKC (144). In cultured mouse PASMCs, exposure to Hx promoted cell proliferation and caused upregulation of PKCδ and PKCε expression and increased phosphorylation of AKT and extracellular signal-regulated kinase (ERK), suggesting a role of novel PKCs and downstream AKT and ERK signaling in Hx-induced PASMC proliferation, and offering new target for molecular therapy of PH (160).

COMBINATION VASODILATOR TREATMENT STRATEGIES IN PH

Because of the complexity of PH pathobiology, a widely adopted treatment strategy is to use a combination vasodilator therapy rather than a monotherapy approach. Much of the current treatment strategies has centered on initial combination therapy with PDE5i tadalafil and ETR antagonist ambrisentan. Sildenafil/ambrisentan combination therapy is more effective than sildenafil monotherapy in improving mPAP in patients with PH (161). Oral PGI₂ analog selexipag may be added as a triple oral combination therapy in case of insufficient response to oral combination therapy with PDE5i and ETR antagonist. However, more is not always better. In a multicenter, double-blind, randomized phase 3b study in newly diagnosed patients with PAH, both initial triple (tadalafil, macitentan, and selexipag) versus initial double (tadalafil, macitentan, and placebo) oral therapy markedly reduced PVR by week 26, with no significant difference between groups (162).

VSMC PHENOTYPIC SWITCHING AND MEDIAL THICKENING IN PH

Although increased vasoconstriction is recognized as a pathophysiological component of PH, only 10%–20% of patients have an adequate vasodilator response when examined by a standardized procedure (163, 164). This suggests that either vasoconstriction is of much lower magnitude than what can be detected by the vasodilator test, or that a fixed component in the pulmonary vascular wall plays a more prominent role in the pathogenesis of PH. One of the hallmarks of PAH is a progressive obliteration of small pulmonary arteries due to exaggerated PASMC proliferation and resistance to apoptosis. This opens new research avenues regarding changes in PASMCs from a contractile to proliferative phenotype and potential loss of the ability of dedifferentiated VSMCs to contract and relax (165). This may also lead to the evolution of PH therapy from vasodilators to antiproliferative drugs (166).

Studies have shown that PAH is associated with remodeling of the small pulmonary arteries, exaggerated vascular cell proliferation, and obliteration of the pulmonary microvasculature (15, 167, 168). We have shown that phenylephrine-induced contraction, and ACh- and sodium nitroprusside-induced relaxation are reduced in pulmonary arteries of MCT, Hx, and Su5416/Hx rat models, suggesting decreased VSMC contractility and reduced responsiveness to vasodilators in PH (68, 84). This could be related to extensive remodeling of the pulmonary arterioles and increased PASMC proliferation. Hx induces VSMC proliferation and migration and promotes synthetic PASMC phenotypic switching (169–171). Loss of PASMC differentiation leads to excessive proliferation and migration and resistance to apoptosis, all of which lead to medial hypertrophy in the pulmonary arterioles. In Hx rat model, hypoxia-inducible factor-1α (HIF-1α) promotes the expression of Cx43, and the HIF-1α/Cx43 axis regulates the proliferation and migration of PASMCs (172). Augmented total and phosphorylated transcription factor CREB and reduced CREB phosphatases are associated with increased PASMC proliferation in MCT and Hx models (173). Increased release of growth factors such as VEGF, platelet-derived growth factor (PDGF), and TGFβ can activate the JAK/STAT pathway and further induce PASMC proliferation, migration, and transformation to synthetic phenotype. The “proliferative” or “synthetic” VSMCs do not contract because they lack the contractile protein myosin and other contractile markers. Cartilage oligomeric matrix protein (COMP) is believed to have protective effects in the cardiovascular system, and serum COMP levels are decreased in patients with PH, especially in the female subgroup, as well as in rat models of PH (174). We have shown increases in wall thickness of pulmonary arterioles of MCT, Hx, and Su5416/Hx models of PH (68, 84), supporting the contention that the decreased pulmonary artery responsiveness to vasoconstrictor and vasodilator stimuli is related to pulmonary vascular remodeling and PASMC switching from a contractile to synthetic/proliferative phenotype.

It is increasingly appreciated that alternative approaches such as antiproliferative, proapoptotic agents could hold promise in further improving the outcome of PH (56–58, 175, 176). ANG II is a major promoter of VSMC proliferation. In a large retrospective cohort of patients with PH, the use of angiotensin-converting enzyme (ACE) inhibitors and ANG II receptor blockers was associated with improved survival. Also, the ANG II receptor blocker losartan reversed PH and right ventricular remodeling induced by chronic inhaled nicotine in rats and mice (177). Sotatercept is a fusion protein that binds activins and growth differentiation factors and could correct the imbalance between growth-promoting and growth-inhibiting pathways in PH (178). In preclinical models of PH, increased PASMC proliferation and pulmonary arterial stiffness are associated with activation of the Hippo pathway and concomitant increase in the expression of STAT-3, Yes-associated protein (YAP), and Notch-3 (179, 180). The prospect of interrupting this mechanobiological feedback loop to restore pulmonary vascular homeostasis offers promise as a novel strategy for treatment of PH.

In addition to their role in vascular relaxation, K⁺ channels are important in many aspects of VSMC homeostasis including the phenotypic switching from a contractile to a synthetic type seen in PH (181). K⁺ channels regulate key cellular processes such as VSMC differentiation, cell cycle control, proliferation, migration, and apoptosis through multiple mechanisms (182, 183). In general, upregulation of K⁺ channel activity leads to antiproliferative and proapoptotic effects, and these effects are often channel isoform-specific (111, 184). Given that different K⁺ channel types have been identified in VSMCs and that they regulate VSMC homeostasis through multiple mechanisms, careful consideration of the channel isoforms, expression levels, and stage of differentiation and activation is needed to optimally leverage this knowledge therapeutically.

We have shown that mild acidification by treating rats with NH₄Cl (1.5%) in the drinking water improved pulmonary hemodynamics, reduced RVSP, right ventricular weight, and pulmonary arteriolar hypertrophic remodeling, and improved PASMC contraction and responsiveness to vasodilators in pulmonary arteries of MCT and Hx models (Fig. 4) (185). Extracellular acidosis inhibits proliferation and migration of cultured rat and mouse VSMCs (186, 187), likely by inhibiting their proliferative and migratory response to growth factors and by increasing their susceptibility to apoptosis (188). Carbonic anhydrases are ubiquitously expressed enzymes that catalyze the hydration of carbon dioxide to bicarbonate and induce mild acidosis. Ongoing studies are evaluating the therapeutic potential of acetazolamide (ACTZ) and other carbonic anhydrase inhibitors in cancer, obesity, allergy, and inflammation (189–191). Interestingly, ACTZ shows antiproliferative properties in cancer cells (192). ACTZ also improves the pulmonary hemodynamics in Hx rat model (193). We have shown that treatment of Su5416/Hx rat model with ACTZ or NH₄Cl induces metabolic acidosis, decreases RVSP and mPAP without affecting left ventricular systolic pressure, and reduces right ventricular hypertrophy (93, 194). Also, tissue histology of peripheral lung sections showed ameliorated pulmonary vascular remodeling and reduced arteriolar wall thickness in ACTZ-treated versus nontreated Su5416/Hx rats. Treatment of Su5416/Hx rats with ACTZ also improved pulmonary artery contraction and ACh- and sodium nitroprusside-induced relaxation, supporting that mild acidification improves PASMC contractility and responsiveness to vasodilators (93). In support, pulmonary arteries harvested from Su5416/Hx rats show a PASMC dedifferentiation pattern with decreased mRNA levels of the contractile markers Myocd (myocardin), Tagln (transgelin), Smtn (smoothelin), and Acta2 and increased proliferative marker Ccnd1 (cyclin D1), and treatment of Su6516/Hx rats with ACTZ partially restored the contractile markers and reduced the proliferative marker (194).

Figure 4.

Potential benefits of mild acidification in pulmonary hypertension (PH). Mild acidification could affect pulmonary endothelial cells leading to increased release of vasodilators and improved relaxation, as well as pulmonary arterial smooth muscle cells (PASMCs) leading to restoration of contractile phenotype, improved response to vasodilators, decreased proliferation and migration. and reduced medial thickening. Mild acidification could also affect components of the extracellular matrix (ECM) leading to decreased fibrosis and hypertrophic remodeling, as well as inflammatory cells leading to decreased macrophage infiltration and reduced release of cytokines. These beneficial effects of mild acidification have improved the hemodynamics and reduced mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), and right ventricular hypertrophy in rat models of PH. NO, nitric oxide; NOS, NO synthase.

PULMONARY ARTERIOLAR FIBROSIS AND REMODELING IN PH

The extracellular matrix (ECM) determines the structural architecture of the pulmonary vasculature and the myocardium and provides mechanical stability necessary for tissue homeostasis. PH is associated with pathological remodeling of the pulmonary vasculature and right ventricle, excessive deposition of structural and nonstructural proteins, and abnormal expression of TGFβ, fibroblast growth factor (FGF), MMPs, and other proteases in ECM. Aberrant TGFβ signaling is linked to PH. BMPR2 mutations perturb the balance between BMP and TGFβ pathways, leading to vascular remodeling and narrowing of the lumen of the pulmonary vasculature (195). Reduced BMP and BMPR2 have also been linked to reduced sphingosine-1-phosphate and zinc transporter ZIP12 signaling in MCT rat model of PH (196). Also, FGF receptors 1 and 2 (FGFR1/2) are elevated in patients with PH and in Hx mouse model (197).

Changes in the amount and content of ECM have been observed in patients with PH and animal models, supporting a role of ECM in disease progression. Accumulation of ECM proteins could also impact vascular cell biology, proliferation, and migration (198). Cardiac magnetic resonance imaging has shown increased pulmonary artery stiffness in PAH (199). Imbalance between MMPs and TIMPs could be an important pathobiological component of PH. MMP-8 is an interstitial collagenase involved in regulating fibrosis and inflammation in the lung and vasculature. MMP-8 expression is increased in plasma and pulmonary arteries of patients with PH compared with controls and in the Hx mouse model. MMP-8^−/− mice show high mortality, increased RVSP, severe right ventricular dysfunction, and exaggerated vascular remodeling, compared with WT mice. MMP-8^−/− PASMCs also show increased proliferation, suggesting protective role of MMP-8 by altering ECM composition in PH (200).

Serum levels of procollagen III NH₂-terminal peptide, carboxyterminal propeptide of type I procollagen, MMP-2, and MMP-9 are decreased in patients with chronic thromboembolic PH compared with healthy subjects. Also, MMP-2-to-TIMP-1 ratio is lower in patients with PH than controls, and correlates negatively with PVR, suggesting that decreased MMP-2 or increased TIMP-1 could be an important factor in pulmonary vascular remodeling (201). Changes in the structure and function of elastin may also have an impact on the proximal pulmonary arterial mechanics in hypertensive calves (202).

Antifibrotics and other molecular and cellular approaches to reduce pulmonary artery remodeling could be useful in PH therapy. Emergent targeted approaches for PH include the development of a TGFβ ligand trap, and upregulation of BMPR2 (196). Angiotensin-converting enzyme 2 (ACE2) catalyzes the hydrolysis of the vasoconstrictor ANG II into the vasodilator Ang-(1–7), and is an important regulator of blood pressure and cardiovascular function. In MCT + Hx rat model of PH, the ACE2 activator diminazene aceturate reduced serum and perivascular focal adhesion kinase (FAK) and improved pulmonary arteriolar remodeling (203). Also, galectin-3 is a carbohydrate-binding lectin that affects reactive oxygen species production, NOX enzyme expression, inflammation, and fibrosis, and in turn contributes to pulmonary artery remodeling and PAH, and downregulating galectin-3 could be useful in PH (204).

Lineage tracing studies have provided important insights into the role of other cell types in the pulmonary vascular wall in the pathogenesis of PH including pericytes and VSM progenitors. Pericytes are mesenchymal-derived mural cells localized in the basement membrane of pulmonary capillaries. Besides structural support, pericytes regulate pulmonary vascular tone and produce ECM components and factors that control vascular homeostasis and angiogenesis. Pericytes may also produce profibrotic and proinflammatory cytokines and in turn contribute to vascular pathology. In the lung, loss of pericyte communication with alveolar capillaries and a switch to a profibrotic/proinflammatory phenotype would promote vascular remodeling, fibrosis, and inflammation in PH (205). Further studies to dissect the complex interactions of pericytes with other pulmonary and vascular cells would help to understand the mechanisms of PH and offer opportunities to develop novel therapeutics.

Resident VSM progenitors at the junction of the muscular to nonmuscular part of the pulmonary arterioles may contribute to pulmonary vascular remodeling in Hx-induced PH via clonal expansion mediated by the pluripotency transcription factor KLF4 (206). Studies in a chronic inflammation model of experimental PH showed that a subpopulation of VSMCs with high levels of Notch-3 expression is the major cellular origin of neointima formation (207).

In addition to resident cells, circulating vascular progenitors and stem cells including endothelial progenitor cells (EPCs), mesenchymal stromal cells (MSCs), and fibrocytes may contribute to vascular remodeling in PH (208). CXCR4 is a stem cell surface receptor of the cytokine CXCL12 that regulates homing of hematopoietic progenitor cells and their mobilization. Bone marrow-derived CXCR4 and proangiogenic cell accumulation could play a role in the development of PAH. CXCR4 expression is increased in pulmonary arteries of patients with PAH and Hx rat model. Downregulation of CXCR4 expression reduced β-catenin levels and PASMC proliferation, and ameliorated pulmonary vascular remodeling in Hx rats, suggesting that CXCL12/CXCR4 is an important factor in PAH (209).

ROLE OF INFLAMMATION IN PH

Irrespective of pathogenic origin, dysregulated immune and inflammatory responses underlie the pathobiology of PH. Human and preclinical studies have suggested a role of inflammation in PH pathogenesis (210–215). A skewed immune response favoring a proinflammatory environment leads to infiltration of inflammatory cells such as lymphocytes, macrophages, and neutrophils. Elevated circulating levels of the proinflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) predicted poor outcomes in idiopathic and familial patients with PAH (213, 216). Also, PAH lungs show characteristic perivascular inflammatory infiltrates comprising lymphocytes, mast cells, dendritic cells, and macrophages (212, 217). Our group has shown induction of IL-6 and CCL2 and upregulation of IL-1β mRNA in lung specimens from human idiopathic PAH (194). Recruitment and activation of proinflammatory M1 and alternatively activated M2 macrophages have been demonstrated in the Hx mouse model and the Su5416 athymic rat model of PAH (214, 218). Studies in MCT rat model showed temporal change of macrophage M1/M2 polarization status in the development of PH, with CD68⁺NOS2⁺ M1-like macrophages participating in the initial stage of inflammation by accelerating apoptosis of endothelial cell, while CD68⁺CD206⁺ M2-like macrophages predominating in the reparative stage of inflammation and the following stage of vascular remodeling (219). The plasma levels of osteopontin, a pleiotropic protein involved in inflammation and fibrogenesis, are elevated and associated with PAH among patients with connective tissue diseases, and may serve as a biomarker for early diagnosis and intervention in PAH (220). TNFα may drive PAH by suppressing tBMPR2 and altering Notch signaling (221). Other studies suggested a role of IL-6/IL-21 signaling in the pathogenesis of PAH (222). IL-6, IL-13, and IL-11 activate the JAK/STAT pathway and induce pulmonary artery remodeling and fibrosis. We and others have shown increased alveolar macrophages and pulmonary expression of proinflammatory mediators Tnf, Il-6, and Ccl2 in Su5416/Hx model of PAH (194, 223). Of note, treatment of VSMCs with TNFα or IL-1β alters cell morphology, increases cell proliferation in response to 20% fetal bovine serum, and results in downregulation of the transcriptional coactivator myocardin (Myocd) and markers of contractile VSMC phenotype including Tagln (transgelin), Smtn (smoothelin), Acta2, Cnn1 (calponin 1), and Myh11 (myosin heavy chain 11), while upregulating markers of dedifferentiation such as Rbp1 (retinol-binding protein 1) and proliferation such as Ccnd1 (cyclin D1) (194, 221, 224, 225).

The identification of inflammation as a key pathogenetic factor in PH has directed research into specific therapies that could ameliorate the inflammatory process. Several proinflammatory pathways have been proposed as novel therapeutic targets for PH. Preclinical studies using anti-inflammatory approaches have shown promise. For instance, pharmacological targeting of TNFα or the IL-6 receptor ameliorates experimental PAH (221, 222). There are also compelling preclinical data to support the benefits of IL-6 blockade using tocilizumab, but the clinical evidence is not as robust. Blocking macrophage leukotriene B4 could prevent endothelial injury and ameliorates experimental PH (226). Immunomodulatory therapies have also been sought to improve the outcome of PAH (59, 175, 227).

Regulatory T cells (Tregs) are important contributors to autoimmunity, and their activity may be compromised during the inflammatory process in PAH. In support, athymic Treg-depleted rats treated with Su5416 develop PAH, which is prevented by infusing missing CD4⁺CD25highFOXP3⁺ Tregs (228), and these effects are exaggerated in female compared with male animals (229).

Immunosuppressive therapy may have some benefits in PH. However, clinical trials of immune modulators in PAH have yielded conflicting results (230), highlighting the need for other approaches. New avenues for targeting inflammation in PH include more targeted anti-inflammatory and proresolution strategies. Targeted expression of HO-1 has been shown to ameliorate the pulmonary inflammatory and vascular responses to Hx (215). In MCT model, BI113823, a selective kinin B1 receptor antagonist reverses experimental PH, decreases macrophage infiltration and TNFα and IL-1β cytokine production, and downregulates MMPs in the lungs (231). Resolvin E1 is a proresolving lipid mediator that could have protective effects in inflammatory diseases. Resolvin E1 levels are reduced in the plasma of patients with idiopathic PAH and in lungs from MCT rat model and Su5416/Hx mouse model of PH. Also, resolvin E1 treatment ameliorated experimental PH and reduced Hx-induced Wnt7a/β-catenin signaling and PASMC proliferation (232).

Mild acidosis could suppress inflammation through different metabolites such as lactic acid, ketone bodies, or protons (H⁺) (233–235). Also, carbonic anhydrase inhibition has anti-inflammatory effects by inhibiting macrophage polarization (194). Treatment with ACTZ or NH₄Cl reduces macrophage infiltration and expression of proinflammatory mediators in Su5416/Hx rat model, and inhibits cytokine-induced dedifferentiation in PASMCs (194).

Studies have shown increases in ACE2 and transmembrane serine protease-2 expression in lung tissue and a decrease in protective sACE2 in patients with chronic obstructive pulmonary disease (COPD), which may represent the possible risk factors for an increased susceptibility of patients with COPD to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (236). COPD and severe PH have been observed as major sequelae after severe postacute COVID-19 pneumonia (237). Also, histological analysis revealed that patients who died of COVID-19 exhibited thickened pulmonary vascular walls, an important feature of PAH (238). Pulmonary thrombosis is common in association with COVID-19, and the risk exists before and during hospitalization, and continues at least up to 4 wk after discharge (239), and its long-term effect on thromboembolic PH needs to be assessed. Interestingly, patients with COVID-19 infection may show a dysregulated acid-base status influenced by carbonic anhydrase activity, and ACTZ may be useful as a pharmacological approach for ameliorating COVID-19 symptoms (240).

CONCLUSIONS AND FUTURE DIRECTIONS

PH is characterized by various degrees of pulmonary vasoconstriction and progressive fibroproliferative obliteration of pulmonary arterioles, pulmonary vascular remodeling, fibrosis, and inflammation, leading to progressive increase in PVR and right ventricular hypertrophy and failure. An imbalance between pulmonary vasodilators including NO and PGI₂ and vasoconstrictor factors such as ET-1 is recognized as an important factor in PH. Therefore, current therapies for PH are focused on vasodilators such as PDE5i, ET_AR antagonists, PGI₂ analogs, and Ca²⁺ channel blockers. To enhance their effectiveness, vasodilators are often used in combination therapy of PH. However, current vasodilator therapies are not universally successful in altering PH progression and increasing survival. Many patients do not respond adequately to vasodilators, largely due to a phenotypic switch in PASMCs from a contractile to a synthetic phenotype, with increased PASMC proliferation, excessive pulmonary vascular remodeling, fibrosis, and inflammation. Interventions to improve the responsiveness of the remodeled pulmonary arteries to vasodilators are emerging as novel strategies in PH. These novel strategies should target pulmonary artery remodeling, fibrosis, and inflammation using new approaches that restore the pulmonary vascular cell phenotype and reduce PASMC proliferation and ECM deposition. Ample preclinical evidence supports that antiproliferative, proapoptotic, immunomodulatory, and cell-based therapies could be effective in PH (56–59, 175, 176, 227, 241); however, translation to clinical application is lagging behind experimental evidence. Induction of mild acidosis could reverse PASMC phenotypic switching, improve the pulmonary hemodynamics and vascular function, and reduce pulmonary artery remodeling, and therefore may provide a complimentary approach to enhance the effectiveness of vasodilator therapy in PH. ACTZ is a well-studied carbonic anhydrase inhibitor and inducer of mild acidosis with multiple clinical applications. Further studies using suitable experimental models of PH and additional methods of controlling and monitoring acidosis in vivo are needed to define the underlying mechanisms and translational potential of these new approaches.

GRANTS

This work was supported by Brigham Research Institute (BRI) Fund to Sustain Research Excellence from BRI (to R. A. Khalil) and National Heart, Lung, and Blood Institute Grants HL111775, R56HL147889, and R01HL147889-A1 (to R. A. Khalil) and R01HL116573 (to H. Christou).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.C. and R.A.K. conceived and designed research; H.C. interpreted results of experiments; H.C. and R.A.K. prepared figures; H.C. and R.A.K. drafted manuscript; H.C. and R.A.K. edited and revised manuscript; H.C. and R.A.K. approved final version of manuscript.