Involvement of TGF-β, mTOR, and inflammatory mediators in aging alterations during myxomatous mitral valve disease in a canine model

Abstract

Inflammaging, a state of chronic low-grade inflammation associated with aging, has been linked to the development and progression of various disorders. Cellular senescence, a state of irreversible growth arrest, is another characteristic of aging that contributes to the pathogenesis of cardiovascular pathology. Senescent cells accumulate in tissues over time and secrete many inflammatory mediators, further exacerbating the inflammatory environment. This senescence-associated secretory phenotype can promote tissue dysfunction and remodeling, ultimately leading to the development of age-related cardiovascular pathologies, such as mitral valve myxomatous degeneration. The species-specific form of canine myxomatous mitral valve disease (MMVD) provides a unique opportunity to investigate the early causes of induction of ECM remodeling in mitral valve leaflets in the human form of MMVD. Studies have shown that in both humans and dogs, the microenvironment of the altered leaflets is inflammatory. More recently, the focus has been on the mechanisms leading to the transformation of resting VICs (qVICs) to myofibroblast-like VICs (aVICs). Cells affected by stress fall into a state of cell cycle arrest and become senescent cells. aVICs, under the influence of TGF-β signaling pathways and the mTOR complex, enhance ECM alteration and accumulation of systemic inflammation. This review aims to create a fresh new view of the complex interaction between aging, inflammation, immunosenescence, and MMVD in a canine model, as the domestic dog is a promising model of human aging and age-related diseases.

Introduction

Cardiovascular diseases, including myxomatous mitral valve disease, are a leading cause of morbidity and mortality worldwide. The pathogenesis of these conditions involves complex interplays between inflammatory processes, cellular senescence, and age-related changes in the cardiovascular system. These factors contribute to the development and progression of various cardiovascular disorders, making them important areas of study for improving disease prevention and management. Myxomatous mitral valve disease (MMVD) affects approximately 2% of the human population and is a significant clinical problem [1]. Consequently, the development of MMVD leads to significant mitral valve regurgitation, left ventricular volume overload, reduced ejection fraction, pulmonary hypertension, and finally, congestive heart failure [1, 2]. The same problem affects other species, particularly dogs. Among them, MMVD occurs with much higher frequency, so the dog represents a useful model for the search for disease mechanisms and also for new therapeutic options (Fig. 1). Some breeds, such as the Cavalier King Charles Spaniel, Dachshunds, and Maltese, are particularly predisposed to develop MMVD [2, 3]. However, extrapolation of the results obtained in dogs should be careful due to the differences in the disease phenotype that can be identified in humans. Dogs do not have mitral valve leaflet atherosclerosis and do not develop a calcification phase in the altered valve apparatus affected by myxomatous degeneration [4–8]. Furthermore, severe fibrosis of the human valves distinguishes human MMVD from canine MMVD [9]. The lack of progression from myxomatous degeneration to the forms seen in humans allows a deeper look into the pathophysiology of this phenomenon in dogs.

Fig. 1.

The photo shows the mitral valve of a 13-year-old German Shepherd dog affected by myxomatous degeneration in each of the leaflet scallops. The valvular apparatus consists of an annulus, two leaflets (anterior and posterior), chordae tendineae, and papillary muscles. The anterior leaflet consists of A1, A2, and A3 scallops, while the posterior leaflet, in turn, consists of P1, P2, and P3 scallops

The common factor for both species is the close relationship between MMVD and age [6, 8, 10]. Aging, being a ubiquitous state of nature in living organisms, is essentially a network of interrelated mechanisms controlling cell differentiation, cell integrity, cell communication, the efficiency of their functions, their ability to produce and use energy, and much more. According to Kennedy et al. (2014), aging is based on 7 pillars: inflammation, stem cell regeneration, macromolecular damage, stress, proteostasis, metabolism, and epigenetics [11]. Findings of age-related changes in valve leaflets indicate increased collagenolytic activity, elastin degradation, and altered metabolism of glycosaminoglycans and proteoglycans [12–17]. These changes are associated with the activity of valvular interstitial cells (VICs) and valvular endothelial cells (VECs) (in particular, VICs that transition from a resting state to an activated myofibroblast phenotype [18–21]), which, under the influence of mechanical stress and aging processes, alter the extracellular matrix (ECM). However, this mechanism is not unidirectional and we know that changes in the ECM environment can also affect cellular activity [22–24]. The mitral valve, given its placement and the function it performs, is subject to constant mechanical stress—changes in pressure between the atrium and ventricle resulting from the different phases of the heart action—as well as physiological stress—cellular senescence, the normal course of which can be disrupted by the previously mentioned mechanical stress. This process is gradual and as yet unstoppable. In humans, the delicate nodular thickening observed at a young age in 30–40-year-olds is already rarely noted in people 65 + , in whom the picture of degeneration is evident [25, 26].

The integration of aging processes is attributed to inflammatory mediators, which are supposed to be a background and indispensable component of age-related diseases [27, 28]. The inflammatory response associated with aging is a sterile, global, low-grade, and chronic inflammation that gradually increases with age, of a different nature compared to inflammation associated with contact with a pathogen or trauma. Age-related chronic inflammation—inflammaging—was put in the spotlight by gerontologists in 2000 by Franceschi [29]. Today, we know that inflammaging is, among other things, the result of the formation of a senescence-associated secretory phenotype (SASP), which is a direct consequence of cellular senescence. According to many authors, global inflammation creates conditions conducive to the development of overt age-related diseases and may also induce VICs to remodel the ECM or promote this process [30]. Given the discoveries in a canine model regarding the accumulation of inflammatory effects over their lifespan [31], the association of MMVD with immune pathways [32], and the association of MMVD pathophysiology with pro-inflammatory pathways and those involved in aging (phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)) [33], it is reasonable to claim that inflammaging and cellular senescence is also important in the development of MMVD in dogs [10]. Recently, research into the inflammatory nature of MMVD development in dogs has been revisited, identifying new signaling pathways deepening the understanding of the pathophysiology of this disease [34] and there has been a significant increase in knowledge of canine mitral leaflet cell senescence. Exploring advances in gerontological research, this review aims to shed light on the complex interplay between aging, inflammation, immunosenescence, and MMVD in the canine model since the domestic dog is a promising model of human aging-related diseases.

MMVD in humans and dogs—comparison and inflammatory background

The domestic dog is a promising model of human aging [35, 36] (Fig. 2). As in humans, senile frailty in dogs derives from impairment of basic functions and impairment of constitutive vital processes [36–39] (Fig. 2), of which cardiorespiratory impairment and the presence of chronic inflammation are among the main and also concurrent conditions [36, 40], where it is often not possible to determine which is causal and which is consequential [41]. Cardiac valvulopathies are one of the most common age-related pathologies in humans. Age-related changes have so far been best studied in the aortic valve [42]. Studies on the mitral valve mainly focus on rheumatic mitral valve disease and the secondary phase, Barlow’s disease [19]. From the perspective of years of research on MMVD in dogs and humans, we know that the changes that occur are progressive and begin in humans around the fourth decade [6–8]. In dogs, it is more breed-dependent, among which MMVD in Cavalier King Charles Spaniel (CKCS) appears earliest (about 6–7 years of age), and also, the progression is most dynamic [43]. In other breeds and hybrids, lesions appear around 9–10 years of age [44]. Some CKCSs were already dying before the age of 10 through MMVD, as a result of lesions whose first symptoms were diagnosed as early as 1–2 years of age [45, 46]. Other breeds live to old age due to slow progression. However, despite their different progression and manifestation, they appear to be the same disease with similar pathophysiology [47]. A predisposition to early cardiovascular disease, which is usually associated with old age (the case of MMVD in some CKCSs), is also a trait in some racial and ethnic groups in humans with atrial fibrillation [48], which are associated with mutations that increase predisposition to, among other things, the development of arrhythmias [49]. It is not excluded that the CKCS predisposition to MMVD derives from the accumulation of multiple gene variants, which, in a way, could explain some of the success of breeding programs limiting the reproduction of individuals with early MMVD [45, 46]. Similarities to humans also exist in the management of the disease and its progression. Due to the primary nature of the mitral regurgitation that develops as a result of MMVD, symptom relief is through the use of diuretics and afterload-reducing drugs [2, 50]. By design, only interventional management can reduce the mitral regurgitation—in both species, transcatheter edge-to-edge repair is used [51, 52]. As in humans, signaling pathways to serotonin are activated in dogs with MMVD [53], no less; there is also a difference, including the lack of association of MMVD in dogs with gene mutations, e.g., Filamin-A, which correlates with the occurrence of the disease in humans [5]. Also of interest is the increased localization of myxomatous lesions in the anterior leaflet (in dogs), or in the posterior leaflet (in humans) [54, 55]. The concretion of myxomatous lesions in humans is also expressed by significant fibrosis that accompanies “overlays” on the surface of the leaflet, which significantly differentiates the dog from the human [54, 55]. The reason for this difference is not precisely known.

Fig. 2.

The main advantages and disadvantages of the dog as an MMVD model are set against characters for rodents

Originally, MMVD was a disease whose development was not associated with inflammation. Subsequent findings have indicated that both the myxomatous mitral valve lesions found in Marfan syndrome (induced in pigs and mice) and those in spontaneous MMVD found in humans and dogs are consistent in terms of the presence of an inflammatory microenvironment [56]. Cytokines, components of inflammatory factors, can act on mitral obturator cells, and induce VICs to remodel the ECM [57]. In a mouse model of Marfan syndrome, inhibition of “Wingless-related integration site” (WNT) pathways inhibited both the progression of valve degeneration and inflammation, demonstrating the link between the two conditions [58]. It, therefore, represents a promising therapeutic target for the early treatment of not only congenital MVD in Marfan syndrome but also acquired MMVD. The definition of inflammaging has brought it into the pantheon of elements of theories about the network nature of aging and associated age-related impairment of the immune system [59]. In essence, inflammaging is the result of physiological aging processes at the cellular and systemic levels. Due to advances in knowledge and the capabilities of veterinary medicine, many more dogs are currently living to a late old age. In highly developed countries, dogs over 10–11 years of age account for 10–15% of the population [60, 61]. At advanced ages, often not predicted by evolution, inflammaging plays a pathological function by interfering with healthy aging and promoting the onset of age-related diseases. The phenomenon of inflammaging is also accompanied by the “two-stroke” hypothesis, according to which inflammatory stimuli sustained over time (first stroke) contribute to the expression of genetic factors (second stroke) predisposing to local diseases, thus giving age-related diseases an “inflammatory background” and “genetic background.” Many breeds, being the result of the intensive influence of breeding based on the desire for a suitable phenotype, have perpetuated sets of genes in their genotype that cause, or progression factor, a predisposition to the development of particular diseases [62], and this also applies to MMVD in dogs [5, 63–65]. It seems unrealistic to comprehensively cover all the mechanisms leading to the creation of an “inflammatory background” and “genetic background” in MMVD because of the complexity and also the intertwining of causality and consequence of the phenomena mentioned. However, its main sources, cellular senescence, and immunosenescence, can certainly be distinguished.

Inflammaging and MMVD in the canine species

The domestic dog, being a model of human inflammaging, is not an exact match for inflammatory changes affecting humans—but it does have key similarities. First, there are changes in the levels of IL-2 (Table 1), which in humans serves as a well-known marker of aging, having a negative association with chronological and biological age [66, 67]. It belongs to the cytokines involved in the type I response produced by T helper 1 cells (Th1) (e.g., IL-2, IL-12, interferon-gamma (IFN-γ)), but this profile is age-dependent and subject to attenuation. However, there is an increase in type II cytokine activity with age, where the cell is mainly involved in T helper 2 cells (Th2) (e.g., IL-4, IL-5, IL-6, IL-10, Il-13) [68–71] (Table 1). Similarly, a reduction in IL-2 levels and a reduction in IL-2 receptor expression in older dogs have been demonstrated in dogs [72]. Furthermore, reductions in receptor mRNA expression have been detected in two of the three forms of the IL-2 receptor, IL-2Rα and IL-2Rγ [73]. Interleukin 6 was recently linked to canine age in the present year, indicating its positive association with age [74]. In previous trials, no change was obtained [75], or statistical differences were obtained only between puppies and the rest of the dog groups (adult, senior, and geriatric) [31]. Equally, in the case of tumor necrosis factor α (TNF-α), it also was only this year that data were obtained confirming its association with age and roles in generating low-grade inflammation [72, 74, 75]. We anticipate that the detectability of inflammaging may be impaired by a study group that was not carefully selected. A sensitive marker of inflammatory response, C-reactive protein (CRP), remained below the limit of detection in studies where IL-6 and TNF-α were shown to be significant, indicating that patients were eliminated from the dog group [74]. However, the role of CRP in inflammaging is debatable, as its association with age has not once been determined [74, 76, 77]. IFN-γ also decreased with age [73], IL-8 increased [74], and an age-related decrease in heat shock protein 70 was reported [76]. However, no age-related differences were indicated in the levels of IL-1 [75] (except for a group of older females, which was characterized by higher IL-1 activity [72]), IL-4, and IL-10 [73]. In the context of the last one, IL-10, a recent study detected its reduced anti-inflammatory activity in older dogs [78]. The presence of inflammation has been demonstrated several times in dogs with advanced MMVD and heart failure [79, 80]. However, it is most likely that this inflammation is due to the severity of the patient’s condition, circulatory disorders, pulmonary hypertension, and hypoxia [81–83], and therefore, does not correspond to inflammaging, which could generate the cellular transformations that lead to MMVD. However, interesting data are presenting increasing inflammation with age, which is responsible for cardiac fibrosis and causes diastolic dysfunction [84, 85]. In dogs, there is echocardiographic evidence of age-related diastolic dysfunction, which is characterized by prolonged isovolumic relaxation time in elderly dogs [86]. Equally importantly, PI3K signaling has been identified as crucial in myocardial fibrosis [87]. Rapamycin, a PI3K/AKT/mTOR inhibitor, can significantly reduce levels of cytokines, including Il-1B [57] at higher doses. So far, this has not been studied in detail in dogs, but it has been indicated that some mTOR inhibitors, such as rapamycin, have a potential reversal effect on cardiac aging and may improve systolic and diastolic parameters [88, 89]. However, genome-wide association studies of blood cells in dogs with MMVD do not indicate a strong link between the disease state and inflammation [62, 90–95]. Serum proteomic studies also did not indicate differences in proteins involved in inflammatory signaling [96–104]. However, locally, an increased inflammatory response has been demonstrated in diseased mitral valve leaflets [105]. A total of 322 up-regulated and 269 down-regulated genes were identified in the valves (including 31 associated with inflammatory response, 42 with immune response, among others: up-regulation of toll-like receptor 4 (TLR4), IL-18 IL-6, toll-like receptor 1 (TLR1), and toll-like receptor 8 (TLR8)) [106], and several changes in the expression of inflammatory factors such as TNF-a and IFN-γ [41]. In addition, the levels of IL-8 and IL-6 were also increased [107, 108], and the number of receptors for IL-1 and IL-10 (Table 1) [108]. These changes may also have prognostic significance, as IL-6 and IL-1b concentrations increased with MMVD progression in dogs [109]. An unexpected finding is the absence of an association between the development of MMVD and levels of IL-33 in dogs, which is a member of the IL-1 family and responds to increased cardiomyocyte damage [110]. In contrast, a strong association of IL-33 and the ratio to its receptor, interleukin 1 receptor-like 1 (ST2), with the development of MMVD and with IL-6 and IL-1β concentrations has recently been confirmed (Table 1) [111]. Increased oxidative stress associated with MMVD has been reported several times [112–114]. The amount of circulating mitochondrial DNA (mtDNA), however, was unexpectedly higher in the heart failure stage, which contrasts with human data, where mtDNA decreases with the severity of oxidative stress [114–116]. In turn, features of oxidative damage have been detected in mitral valve cells [33, 117]. Although the most comprehensive recent studies have not indicated an effect of body condition score (BCS) on levels of inflammatory mediators [74], anti-aging strategies based on the administration of substances affecting cellular metabolism (e.g., rapamycin) through calorie-restrictive dietary interventions or diets enriched with anti-inflammatory and antioxidant substances are having an effect by reducing levels of key inflammatory mediators [118–124]. For example, decreases in IL-6 and TNF-α have been reported [125]. Changes indicative of low-grade inflammation may be alterations in platelet levels [126, 127]. Platelet levels in aging dogs decrease [74]; danger/damage associated molecular patterns (DAMPs) (factors mediating sterile low-grade inflammation [128]), increase; one DAMP detected in age-related dogs is fibrinogen [127].

Table 1.

Comparison of several cytokine’s activities in MMVD in dogs and humans. ↑—increase in MMVD, ↓—decrease in MMVD, ±—no statistical change

MMVD in dog	MMVD in humans
IL-7 ↓ [129]	IL-7 ↑ [130]
IL-33 ± [110]	IL-33 ↑ [111]
TNF-α↑ [109]	TNF-α ↑ [131]
IL-1β ↑ [109]	IL-1β↑[111]
IL-6 ↑ [109]	IL-6 ↑[111]
IL-10 ± [132]	IL-10↓ [133]

Cellular senescence: telomere attrition, mitochondrial aging, and DNA damage response

Recently, the mechanisms involved in canine aging per se and cellular senescence have been investigated, and biomarkers of aging have been presented [134, 135]. It should be emphasized that cellular senescence is an ever-present process and allows the elimination of senescent cells (SnCs) and dysfunctional cells, thus protecting the body from aging [136, 137]. Cellular senescence is a sequence of activated antiproliferative programs, and senescence triggers have been indicated by telomere shortening, mitochondrial dysfunction, and higher levels of reactive oxygen species (ROS) as well as expression of certain oncogenes or loss of tumor suppressor genes [134, 135, 138]. Triggers usually interact continuously, but their identification has made it possible to detail the types of cellular senescence. The recipient of the effects of aging triggers is the DNA damage response (DDR), which responds to double-strand breaks (DSBs), which leads to cell cycle arrest [139].

Telomere attrition

We distinguish between replicative senescence (RS), which provides a limited cell lifespan, through telomere (TL) shortening and the activation of programmed cell death processes [138]. Lack of TL protection, resulting from low telomerase (TERT) expression, leads to TL loss during divisions and induces DNA damage, hence the conclusion of the determinant importance of starting TL length for lifespan across species [140]. Healthy canine cells and tissues have similar levels of TERT expression, so comparable TL length measurements can be expected. This was confirmed in dogs by comparing the TL of mononuclear cells in saliva and blood [141], but differences were found in the comparison between leukocytes, adipocytes, and myocytes [142]. This may suggest different aging bluntness between tissues, as mesenchymal stem cells collected from a single canine donor did not differ in TL length [143]. Most interestingly, the TL lengths of mesenchymal cells taken from different large breeds also did not differ significantly from each other (among Border Collie, German Shepherd, Labrador, Malinois, Golden Retriever, and Hovawart, German Shepherd dogs had the shortest TL) [144]. The comparison of large breeds with small breeds indicated longer TL in smaller breeds, which correlated positively with their lifespan and may explain the differences in average chronological age attained among different breeds [145]. However, a common feature is that shorter TL in dogs correlates with lifespan [146, 147] (especially when TL from more than one tissue is taken into account [146]). A common relationship was a predisposition to cardiovascular disease, tumorigenesis [145, 148], and shorter TL have also been correlated with lower canine gamete competence [149, 150]. However, the etiology of MMVD is mainly associated with smaller breeds, and therefore statistically, MMVD should be associated with longer TL, especially if dogs with the initial phase of the disease were studied. However, relevant data are deficient. Tumor cells show increased TERT expression [151, 152], which has become one of the targets of anticancer immunotherapies using adenoviruses to induce responses against dogTERT [153–156]. Relatively high expression of dogTERT is maintained by mesenchymal cells, and there may be differences depending on their origin. Cells derived from bone marrow showed the highest activity compared to skin and adipose tissue cells. Interestingly, this was not reflected in TL length, which was comparable between groups [143]. TERT activity in cardiac tissue is high in newborns, but peaks in TERT activity are also recorded in cardiac pathologies [157]. DogTERT is also an enzyme with increased activity in heart failure in dogs. Tachycardia-induced heart failure and cardiomyocyte death also correlated positively with the number of proliferating myocytes expressing increased dogTERT and Ki67 protein [158]. Increased expression of dogTERT led to TL preservation in subsequent cell divisions of canine cardiac myocytes [158].

The other mechanism of TL protection and regulation in dogs, namely the telosomes, better known as the shelterin, has received limited comment. Shelterin is composed of six protein subunits: telomere repeat binding factor 1 (TRF1), telomere repeat binding factor 2 (TRF2), repressor/activator protein 1 (RAP1), protection of telomere 1 (POT1), TRF1- and TRF2-interacting nuclear protein 2 (TIN2), and TPP1, which are located at the ends of chromosomes [159, 160]. As TL have replication- or oxidative stress-induced DSBs, they can affect DDR activation [161, 162]. Eukaryotic DNA is protected by the shelterin sequence, which insulates TL from DDR reactions [162, 163]. This particular protection, however, interferes with repair processes and contributes to the persistence of DDR [164]. In dogs, increased expression of TRF2, POT1, and TIN2, decreased expression of RAP1, and no change in TRF1 and TPP1 expression were detected in spermatozoa with shorter TL [149]. Shelterin protects against serine/threonine signaling (ATM), among other things. ATM activated as a result of DSBs is an important modulator of cellular senescence, a DDR intermediate, and one of the switches between cellular senescence and apoptosis [165]. Reduced ATM expression levels have been detected in dogs with mammary gland cancer, which may explain their resistance to apoptotic pathways [166]. Most of the genes activated during TL shortening are the result of DDR activation. However, it has been shown that there is a pool of genes that, under the influence of shorter TL, before DDR activation, such as the ISG15 gene, can provoke an inflammatory response and generate inflammaging [167]. Down-regulation of this gene has been detected in stage B1 MMVD in dogs [168]. ISG15 has roles in inflammatory and DNA damage-related responses in the heart and may have a potential link to the development of cardiomyopathy and age-related cardiac disease in humans [169–171]. Increased interest in this gene may be an opportunity for further research in dogs [170].

RS, being an indicator of depletion of proliferative potential, has a framework that is referred to as the Hayflick limit [172]. The human cell, having an intrinsic limitation, has a maximum number of cell divisions, which in humans is about 45–55 divisions, whereas in dogs, due to considerable breed disparity and scarcity of studies, there is no such specific guideline [172]. In a single study on canine mesenchymal cells, a significant slowing of the cell cycle in Border Collie was detected after 45 days (cell division took longer than 100 h). In contrast, other breeds reached the same level after only 25 days [144]. “Canine Hayflick’s limit” in the German Shepherd and Hovawart was 16 and 17 population doublings each. In the Border Collie, 25, and in the Golden Retriever, Labrador, and Malinois, 20 doublings were achieved [144]. However, the maximum number of divisions may be reduced by complications of normal RS cellular senescence.

Mitochondrial dysfunction

Stress-induced premature senescence (SIPS) is a sub-cytotoxic stress that induces DNA damage, in which mitochondrial dysfunction plays a major role and is mediated by ROS of mitochondrial or exogenous origin [138]. During SIPS, it is not necessary to reach TL that is too short to activate the DDR pathway, which leads to cell cycle arrest [173]. Mitochondria are an energy center and major source of ROS; stabilizing the cellular oxidative status has become key in the eyes of researchers for understanding oxi-inflammaging theory [174–176], especially in light of data on their interaction with the canonical elements senescence and aging; DDR, TL shortening, and immunosenescence [177–179]. Mitochondrial aging is not a well-recognized change, but it is known to contribute to a reduction in the amount of mitochondrial in the cell; there is a decrease in adenosine triphosphate (ATP) production [159], ROS production increases, and mitochondrial membrane potential weakens [180]. Mitochondrial dysfunction has been detected in dogs with MMVD, using the determination of mitochondrial activity in peripheral blood mononuclear cells (PBMC) [181]. Similar to mitochondrial aging, a decrease in ATP production was detected in canine MMVD, but interestingly glycolysis was not altered [181]. Following this, increased mitochondrial damage was noted from the analysis of serum mtDNA levels in dogs with early MMVD [114]. Abnormalities in mitochondrial biogenesis and autophagy result in a reduction in their overall mass, while increased ROS production may result from a compensatory increase in NLRP3 inflammasome activity, either age-related or in response to a stress stimulus [180]. In dogs, changes in ROS and the regulation of mitochondrial metabolism in individual breeds are as yet unclear, to say the least, and therefore require further investigation [182–186]. However, increased values of 8-F2α-isoprostane, a deleterious product of lipid oxidation against a background of oxidative stress, are noted in dogs with MMVD [187].

Dissipation of the mitochondrial membrane potential is associated with impaired respiratory chain function [180], including through proton leakage [182, 188, 189]. Interestingly, there is little change in enzyme activity over the course of age; thus, for pyruvate kinase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase, researchers have not observed differences [186]. Reduction of senescence processes is partly possible with antioxidant therapies. In the aging dog population, the use of antioxidants has been shown to increase ATP production, mitochondrial mass, and cytochrome c oxidoreductase activity [190, 191]. Dogs with MMVD, which show a decrease in cellular respiration [181], could also benefit from the use of antioxidants. This is because the mitochondrial antioxidant capacity, which determines the ability to maintain a redox balance, is reduced. In patients, the antioxidant capacity of copper reduction (CUPRAC) and the equivalent antioxidant capacity of trolox (TEAC) decrease with the progression of MMVD [192].

During life, there are dynamic changes between anabolic and catabolic processes and it is the mitochondrion’s task to maintain an adequate energy balance [193, 194]. Nutrient-sensitive pathways and changes in energy status are currently the main targets of gerontologists in the pursuit of mammalian longevity. Two pathways subject to insulin signaling, the glucose-responsive insulin-like growth factor 1 (IGF-1) and the protein homeostasis-regulating mTOR complex, are also involved in anabolic signaling. On the other hand, 5ʹAMP-activated protein kinase (AMPK) and sirtuins are responsible for catabolism and deficiency signaling. Because of reports from studies in a mouse model of longevity induced by a starvation phenotype (via caloric restriction or pharmacological acquisition of the phenotype) [195–197], suppression of anabolic processes (e.g., rapamycin) and stimulation of catabolic ones (e.g., metformin, resveratrol) are assumed to be anti-aging strategies [198]. AMPK signaling has recently gained importance in signaling cardiac metabolism in a canine model of heart failure and atrial fibrillation. It has been indicated that it reduces lipid accumulation via AMPK/peroxisome proliferator-activated receptor-α (PPAR-α)/very-long chain acyl-CoA dehydrogenase (VLCAD) [199], reduces cardiomyocyte apoptosis, and prevents the progression of heart failure in dogs [200]. However, no effect of AMPK stimulation by metformin has been demonstrated in in vitro studies of young and old dog fibroblasts [201]. With age, a decrease in levels of the coenzyme nicotinamide adenine dinucleotide (NAD +), which affects mitochondrial maintenance, is noted in humans and is crucial, especially in tissues with high energy requirements, including the cardiovascular system [202, 203]. It has been noted that large breeds have higher concentrations of NAD + compared to smaller breeds [204]. Furthermore, it has been indicated that older dogs have higher NAD + concentrations than younger dogs [204]. Increased nicotinamide adenine dinucleotide phosphate (NADPH) activity (a marker of nitric oxide synthase (NOS) [205]) in dogs with MMVD may be indicative of increased mitochondrial oxidative metabolism, which in age-related changes leads to a redox imbalance. Sirtuin1 (SIRT1), involved in the anti-inflammatory and antioxidant response, is an (NAD +)-dependent histone deacetylase involved in the regulation of mitochondrial activity and biogenesis [206]. It also has a close association with p53, forkhead box protein O (FOXO), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α—main regulator of mitochondrial biogenesis) [206]. SIRT1 has also been extensively studied for its high potential for anti-aging activity [207]. Its levels in dogs have been shown to decrease with age [208] in both large and small breeds. One strategy against this trend is the use of activators of SIRT1 expression, namely resveratrol. In dogs, the anti-aging effect of resveratrol is still only being investigated, and the first tests have been carried out on fibroblasts, tumor cell lines, cohorts of healthy dogs, or as part of adjuvant therapy for cancer patients [201, 209–213]. In addition to the known main molecular target of resveratrol, it also has numerous interactions with other members of the sirtuin family [214]. Heart failure with preserved ejection fraction (HFpEF), related to age and inflammaging [215], is a form of failure caused by reduced left ventricular compliance. One of its causes is the progressive expansion of ECM components, as well as an energy imbalance, including the loss of crucial proteins, including sirtuins (1–7) [216, 217]. The anti-aging and anti-fibrotic activity of sirtuins provided by the regulation of SIRT1/Smad3 (suppression of transforming growth factor-β1 (TGF-β1)) [218], SIRT2/LKB1 (activation of AMPK) [219], and SIRT3/H3K27me3 (inhibition of the FOS/AP-1 pathway) [220] contributes to the prophylactic effects of resveratrol, including against HFpEF, reducing cardiac fibrosis in a rat model [221]. Resveratrol activating SIRT1 suppresses pro-inflammatory pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and NLRP3 [222, 223]. SIRT1 is also expressed in canine PBMC in six different variants, in lymphocytes, monocytes, and granulocytes [224], which may explain the effect of resveratrol on the cytokine response and the oxidative nature of the inflammatory cell response to mitogen [210, 225]. Unfortunately, in dogs, few studies have been devoted to the sirtuin family, which all involve SIRT1. Anabolic signaling pathways will be discussed later in another section.

DNA damage response

As a result of elevated oxidative stress, age-related accumulation of DNA damage biomarkers, including 8-oxo guanine (8OHdG), a guanine oxidation product [76, 182, 226, 227], is detected in the blood as a result of cellular senescence (TL shortening, mitochondrial dysfunction, and exogenous ROS stress). Nucleotide damage prevents the propagation of replication forks. DDR, which have checkpoints in the interphase of the cell cycle, control DNA repair mechanisms and when they are impaired [228], or when multifocal DNA damage is detected, the cell cycle is arrested in the G1 or G2 phase [136, 229, 230]. In human DDR, DNA damage is detected by MRN (Mre11-Rad50-Nbs1) and PARP1 (poly (ADP-ribose) polymerase), which has also been recorded in dogs [138, 162, 231, 232]. Recognition of DNA damage contributes to a cascade of events and phosphorylation of ATM, ATR (ataxia-telangiectasia and Rad3-related), and p53 protein, among others [233]. Higher levels of p53 protein in older dogs and those with tumors have been reported on several occasions [234], and it is also elevated due to UV-induced DNA damage [204]. P53 up-regulates the cyclin-dependent kinase inhibitor p21, inducing cell cycle arrest through CDK2 inhibition. The up-regulation of p21 in SnCs has been studied several times, but the results are not consistent [234–236]. An alternative second DDR for cell cycle arrest is p16 signaling, which, through CDK4 and CDK6 inhibition, induces cell cycle arrest by inhibiting retinoblastoma protein hyperphosphorylation [237]. In dogs, higher p16 activity has been indicated when canine fibroblasts are induced to age by Vorinostat [234]. A further component of the cascade whose activity is used as a biomarker of aging is yH2AX. A DNA damage-associated increase in histone yH2AX phosphorylation has been detected in dogs [235], but this has not been confirmed in studies of hepatitis and canine ocular age-related lesions [236, 238]. Increased phosphorylation of yH2AX contributes to the activation of DNA repair processes. In one study dedicated to DDR analysis of mitral valve leaflets of dogs with MMVD, aVICs were shown to be SnCs [33]. Activation of the canonical DDR pathway was detected, with upregulation of p53, P21, p16INK4A, and yH2AX. The analysis of the miRNA profile in MMVD presents interesting results. Researchers confirmed increased expression of p21 protein, for evidence of the activity of pathways leading to senescence, and there was also a decrease in miR-20a and miR-17, characteristic of aging [239, 240]. Hence, the development of the aVIC phenotype may therefore be the result of progressive cellular senescence processes [241]. Moreover, cells with the aVIC phenotype have a reduced proliferative potential, and a significant proportion of them are arrested in G1, S or G2/M phase [33].

Lysosomal activity increases with age and is a response to increased demand for hydrolytic activity [242, 243]. A widespread marker of aging is the enzymatic activity of β-galactosidase (SA-β-gal) [244], which results from an increase in lysosomal activity and is characteristic of SnCs [245]. Increased levels of SA-β-gal were detected in canine fibroblasts subjected to aging with Vorinostat [234]. In contrast, in MMVD, aVICs were significantly more frequently SA-β-gal-positive compared to qVICs, indicative of their senescence [33, 240]. Interestingly, breed differences were identified by comparing several canine mesenchymal cell lines. Based on SA-β-gal concentrations, SnC formation was shown to be twice as fast in German Shepherds, Labradors, and Golden Retrievers compared, for example, to Border Collies [144]. Senescence detection based on SA-β-gal relies on the determination of the enzymatic activity of galactose and is therefore dedicated to fresh biological material or in vitro cultures, whereas in the case of collected clinical material for the detection of SnC cells, staining based on reaction with metabolites of, e.g., oxidized lipids, i.e., lipofuscin, gives much better results [246]. In the case of material as difficult to access as mitral valve leaflets, the possibility to study SnCs in banked material appears to be advantageous. Lipofuscin staining has been used to detect SnCs in canine neurodegenerative diseases over 10 years of age [247].

Cell cycle arrest and SASP

Cell cycle arrest does not ensure programmed cell death, as anti-apoptotic pathways (SCAPs) (PI3K/AKT, p53, BCL-2 family, heat shock proteins (HSPs)) effectively protect SnCs from death [248, 249]. Similarly, aVICs show resistance to apoptosis and undergo numerous changes in the process of acquiring the aVIC phenotype. The altered cells are characterized by increased expression of alpha-actin-2 (α-SMA) and decreased expression of vimentin [240, 250], and their number increased with disease severity [126, 251]. α-SMA is a specific marker of activated myofibroblasts, confirming the altered phenotype of qVICs [19]. In addition, an increase in the key qVIC phenotype change genes Notch, ACTA2 (α-SMA), and MYH10 (Smemb) has been demonstrated [33, 47, 126, 251, 252], which may suggest the influence of transcription factors such as TGF-β [94]. α-SMA^high+ aVICs, while strongly influenced by TGF-β, also show reductions in the expression of miR-20a, miR-17, and let-7c, factors associated with aging with fibroblastic cell transformation [239, 240]. Interestingly, mi-RNAs have been shown to play a key role in the transit of qVICs to aVICs via the miR-145-KLF4-αSMA axis [253]. The plasticity of fibroblasts, and their associated susceptibility to cell mediators, make them an important component of aging and degenerative diseases [254]. The trigger for ROS-mediated fibroblast differentiation is the advanced glycation end-product (AGE). AGE, due to its sensitivity to ROS and its effects on fibroblasts, has been identified as an important factor in aging and the development of age-related diseases [255]. However, in the only study on dogs, AGE association with age and size of the animal was not demonstrated [256]. Fibroblasts are particularly sensitive to the effects of ROS, and oxidative stress, which can accelerate aging processes in fibroblasts. However, fibroblasts are also altered by TL shortening [257]. Numerous resistance pathways lead to the accumulation of aVICs in valve tissue [126, 258]. One apoptotic pathway is caspase signaling. In aVICs of mitral valve leaflets with degeneration, the number of cells positively stained for cleaved caspase-3 was significantly reduced compared to healthy tissue [259]. In addition, a reduced severity of DNA fragmentation and the presence of apoptotic bodies was observed in aVICs compared to qVICs suggesting resistance to apoptosis [259, 260]. Furthermore, an imbalance between pro-apoptotic (BAX) and anti-apoptotic (Bcl-2) pathways has been demonstrated in myxoid mitral valves [260] and altered expression of miR-30d, a miRNA known to regulate apoptosis and regulate myocardial fibrosis [240, 261, 262]. Reduced expression of ATG7, a gene responsible for regulating autophagy, was detected in canine aVICs. Reduced autophagy flux is characteristic of SnCs and leads to the maintenance of dysfunctional organelles, which are a source of, for example, ROS [263]. In contrast, silencing mTOR signaling in aVICs through manipulation of PI3K signaling or selective knockdown of p70 S6K reverses the cell phenotype [33]. Whereas the use of mTOR inhibitors inhibits smooth muscle cell proliferation in pulmonary hypertension [264]. A strong effect of mTOR on α-SMA + cell metabolism creates a new field for pharmacological treatments in heart disease [265]. An example is feline hypertrophic cardiomyopathy (HCM), which, like MMVD and senile cardiac fibrosis, is characterized by abnormalities in connective tissue metabolism. The use of rapamycin in cats with HCM produced a dose-dependent suppressive effect on myocardial hypertrophy and stimulated autophagy [266].

As is well known, SnCs do not remain quiescent; on the contrary, a cell undergoing senescence processes, regardless of the cause, enlarges [267, 268] and is hyperactive and hyperfunctional [269]. Pro-proliferative, pro-growth, and differentiation-stimulating signaling pathways assume the role of the mechanisms driving these changes. Increased PI3K/AKT/mTOR/p70 S6K signaling has been detected in canine mitral valve lesion cells [33]. Long-term activation of the mTOR pathway leads to the accumulation and secretion of cytokines, growth factors, chemokines, ECM-modifying enzymes, and ROS, conferring a SASP to the cell [270], and this process is called geroconversion [271]. Furthermore, it has been shown that aging fibroblasts can transmit senescence signals to the environment, similar to hypersecreting SnCs, which interact in a paracrine manner to alter the tissue environment [173, 272–274], so SASP is thought to be the most important feature of SnCs. α-SMA^high+ aVICs also induced increased gene expression of IL6, and IL18 [107], which is a characteristic set of SASP-secreted factors. The altered activity of aVICs in canine MMVD has been relatively well described in the literature. In addition to the cytokine alterations previously discussed, a large group of up-regulated genes and proteins are the matrix metalloproteinases (MMP): MMP1[275–277], MMP2 [278], MMP3 [279], MMP8 [96], MMP9 [96, 277, 280], MMP12 [41, 106], MMP14 [275, 276, 278], ADAMTS [106, 107]. Some of these such as MMP9 correlated with changes in echocardiographic assessment of lesions in dogs with MMVD [281]. A hallmark of SnCs is the concomitant increase in antagonistic processes (such as mTOR activity and lysosomal activity), which is why increased amounts of tissue inhibitors of metalloproteinases (TIMPs) are shown in the mitral valve leaflet tissue of dogs with MMVD. Among these, TIMP1 [96, 108], TIMP2 [275, 276, 278], TIMP3 [275, 276], and TIMP4 [276] are most abundant. A potent modulator of the ECM is TGF-β, whose abnormal metabolism can lead to severe disorders of connective tissue structure. In severe cases, genetic mutations cause systemic malformations, as in Marfan syndrome [282]. Its strong position among the pathomechanisms of MMVD in dogs has long been postulated. Its particular forms have been identified in increased amounts in diseased mitral valve leaflets, particularly TGF-β1 [41, 278, 283]. TGF-β1 is also particularly important for aging and for cellular senescence [284]. Among other things, TGF-β1 has been suggested to play a key role in aging and cellular accumulation in idiopathic pulmonary fibrosis [285]. In dogs, researchers have recently been able to link TGF-β1 signaling to the aging of mitral valve leaflet VICs [33, 117] and propose its signaling pathways to be central to the pathogenesis of MMVD [286]. Due to the presence of TGF-β1 receptors on the surface of VICs and the excessive production of TGF-β1 by these cells, autocrine TGF-β1 signaling is potentiated and senescence features are spread. TGF-β1-treated qVICs differentiate into aVICs [33], acquiring α-SMApositivity through this signaling pathway [287]. A similar response was obtained in human cells in which TGF-β1 induced qVIC differentiation via Smad3/NADPH oxidase 4(NOX4)/ROS signaling [257]. The canonical TGF-β1 signaling pathway is the induction of phosphorylation of a group of SMAD proteins by contact with the TGF-β1 receptor, leading to activation of transcription factors and changes in the ECM [288]. TGF-β1, however, has alternative signaling pathways by which it engages in PI3K/AKT pathways [33, 117]. It has been indicated that VIC senescence in canine mitral valves may be mediated by both of these pathways [33, 117]. Another potential source of aVICs is mitral valve leaflet endothelial cells that have undergone endothelial-mesenchymal transition (EndoMT) processes [250] (Fig. 3). Abundant evidence was presented indicating damage to the basement membrane of the valve endothelium and also reduced intercellular connections. Increased expression of ACTA2, CTNNB1, SNAI1, and HAS2 and decreased CDH5 (VE-cadherin) expression were noted [107]. VEC cells cultured in vitro several times demonstrated the potential to EndoMT and adopt the phenotype of an active myofibroblast. The simultaneous presence of VE-cadherin in a-SAMpositive cells indicates an endothelial origin [107]. Mitral valve leaflet cells sorted for myxomatous lesion severity showed an increase in endothelial-derived myofibroblasts with disease severity [41]. One theory is that depleting VICs are replaced by EndoMT-derived VECs. Cultured VECs had increased expression of SMemb—a characteristic factor secreted during embryonic development—whose activity leads to the formation of mesenchymal cells from cardiac endothelial cells [250]. Importantly, EndoMT can be induced by both TGF-β1 signaling, followed by Smad regulatory pathways, and by cytokines [106]. It is suspected that EndoMT may be a process secondary to the disease course and may be the result of increased oxidative damage. This would be consistent with a TGF-β1/Smad3/NOX4/ROS activation pathway involving increased production of highly efficient oxidase NOX4 [289].

Fig. 3.

The histological structure of both mitral valve leaflets is identical and consists of three layers: atrial, spongy, and ventricular. The layer facing the atrium is predominantly composed of valvular endothelial cells (VEC). The spongy layer contains loosely arranged collagen and is rich in glycosaminoglycans and fibroblast-like interstitial cells (VICs). The layer facing the ventricle is also covered by VEC, but its basal layer contains more collagen fibers. Two types of VICs can be found in the spongy layer: quiescent VIC (qVIC) and active VIC (aVIC). In dogs as in humans, qVICs are α-SMA^low+ and vimentin ^high+ cells. In contrast, aVICs are α-SMA^high+ and gradually lose the presence of vimentin (myofibroblast-like phenotype). aVICs have a much higher secretory potential compared to qVICs and are responsible for ECM reorganization. Another source of active myofibroblasts is the endothelial-mesenchymal transition (EndoMT), which VEC cells are subjected. EndoMT-derived myofibroblasts are characterized by the simultaneous presence in their structure of VE-cadherins (endothelial-specific adhesion molecules) and α-SMA

In the case of mTOR signaling, the canonical signaling crucial for SASP is the interaction with insulin-dependent receptors. A breakthrough discovery was the identification of a determinant role of IGF-1 on the lifespan of dogs of different breeds [290]. A study by Sutter et al. (2008) identified single IGF-1 nucleotide polymorphisms selectively present only in miniature breeds or only in giant breeds [290]. Furthermore, IGF-1 concentrations, according to cross-sectional studies, are highest among giant breeds, which on average live shorter [291]. In dogs, IGF-1 levels in relation to age have a similar trend to humans and are constitutively negatively correlated with it, also among other mammals [292–294] and also in dogs with MMVD [295]. IGF-1, a gero-gene, is also strongly associated with mTOR activity. Rapamycin treatment of fibroblasts collected from large- and small-breed puppies induced increases in non-glycolytic acidification rates, oxygen consumption rates (OCR), proton leakage, and non-mitochondrial respiration [201]. Dogs receiving 30 days of therapy against graft rejection had increased glucose clearance [296]. Furthermore, insulin release after glucose infusion was higher during rapamycin treatment than before [296]. Similar to the effect of rapamycin on mTOR, the action is induced by calorie restriction (CR) [297]. CR, in addition, slows down aging and prevents age-related diseases. A key interaction between mTOR and IGF-1 is confirmed when persistent starvation leads to starvation diabetes [297]. Together, mTOR and IGF-1 signaling is responsible for the regulation of cell proliferation and differentiation and, in the case of cycle-arrested cells, also co-optimize their geroconversion [298]. The GH/IGF-1 axis is one of the crucial signaling pathways in cell proliferation, differentiation, and aging. Pituitary growth hormone (GH) signaling via the GH receptor leads to activation of janus kinase 2 (JAK2) and phosphorylation of the transcription factor signal transducer and activator of transcription (STAT), which induces IGF-1 production [299]. IGF-1 in SnCs is one of the factors secreted at increased levels [33] and stimulates insulin-receptor substrate (IRS) phosphorylation via the IGF-1R receptor, leading to mTOR activation. Increased IGF-1/IRS/mTOR signaling has been detected in diseased canine mitral valve cells [33] (Fig. 4).

Fig. 4.

The diagram shows the differentiation over time of mitral valvular interstitial cells (MVICs) (from qVICs to aVICs). The diagram includes processes leading to DNA damage and induction of double-strand breaks (DSBs), which induces a DNA damage response (DDR). The p53/p21 and p16 signaling pathways lead to the inhibition of retinoblastoma protein (RB) hyperphosphorylation and induction of cell cycle arrest. Aging cells possess resistance mechanisms against apoptosis (SCAPs), so apoptotic bodies are not formed, and cell death does not occur. aVICs arrested in G1 or G2 phase develop a senescence-associated secretory phenotype (SASP), which is characterized by cell enlargement and a secretory phenotype. Factors secreted by hyperactive cells (MMPs, interleukins, TGF-β) model the ECM and lead to myxomatous degeneration of the mitral valve

Immunosenescence in dogs

The role of the immune system is not limited to generating a response to infectious agents. It is one of the main systems, along with the nervous and endocrine systems, responsible for hormonal and metabolic signaling control. Over the course of life, the immune system, from young adulthood to old age, undergoes numerous changes that affect its functioning [300]. Immunosenescence, or aging of the immune system, is a process present at the organ level as well as in cellular signaling pathways [301]. Although complete knowledge of the mechanisms of immunosenescence is still a mystery to us, several have been identified: thymic involution, reduced production and depletion of naïve cells, altered ratios in immune cell subpopulations (especially T cells), reduced quality of the adaptive immune cell response to antigen (cellular as well as humoral), hematopoietic stem cell (HSC) dysfunction, senescence changes in immune component cells [302], impaired removal of SnCs [303, 304].

Thymus involution

Thymus involution, which progresses with age, is one of the main features of immunosenescence, which in large part may be dictated by evolutionary and also environmental changes and is the first manifestation of immunosenescence [305, 306]. Thymic involution is a constant feature of mammalian immunosenescence and has been linked to age and the occurrence of age-related diseases and chronic inflammation in dogs, cats, and humans [28, 29, 307–312]. The timing of its onset varies between species. In humans, involution progresses from the first year of life [307]; in dogs, the first changes appear in the first months of life [313, 314]. Thymic involution is regulated and results from a reduction in the number of stroma cells and a decrease in their productivity [315]. The reduced competence of the stroma cells to synthesize catalase, resulting in low resistance to oxidative stress, is responsible for the phenomenon of atrophy of such an important organ [316]. From human and mouse studies, we know that the genes and growth factors that are responsible for controlling thymic aging are as follows [315]: Foxn1 gene—belongs to a group of genes responsible in dogs for lymphocyte development whose differential expression is associated with tumorigenesis [317]; WNT—a factor involved in cell growth and differentiation [125]; microRNA [318, 319]; growth factors (e.g,. VEGF)—responsible for thymocyte differentiation and T lymphocyte maturation [320]; TOX gene (thymocyte selection associated high mobility group box)—responsible for regulation of adaptive immunity (T and NK cell differentiation and selection) [125]; CCRL2 (C–C motif chemokine receptor like 2)—involved in G protein-coupled receptor signaling pathway by which it is involved in immune and inflammatory responses [125]. A molecular marker of thymopoiesis and naive T cells is signal joint T cell receptor excision circles (sjTRECs), which are reported to decrease with age [321]. Based on the measurement of sjTRECs in dogs, two phases of decline in thymic function have been noted: between 1 and 5 years of age, and after reaching 9 years of age [321].

T cells

T cells have been extensively studied for their involvement in cardiovascular disease and may contribute to inflammatory cardiac remodeling [322]. T thymic involution contributes to a decrease in the peripheral lymphocyte count, as well as a reduction in the number of naïve T cells [72, 323]. In addition, their capacity for lymphoproliferative responses decreases with age as a result of mitogen exposure [72, 75, 323–330]. The decrease in the number of T lymphocytes, however, is not proportional among the different subpopulations. An age-related change that has been repeatedly confirmed is a disequilibrium in the CD4 + :CD8 + ratio, which results from an increase in CD8 + lymphocytes and a decrease in CD4 + [127, 326, 328, 329, 331, 332]. Fewer data suggest that only the CD8 + population is affected by the changes [333]. According to others, the reduction in the CD4 + :CD8 + ratio is due to different rates of loss of individual lymphocytes, among which the CD4 + subpopulation suffers a dramatic loss in numbers [323, 334]. However, using more accurate methods to detect T cell population phenotypes, dynamic changes in abundance between naïve (TN), central memory (TCM), effector memory (TEM), and TEMRA (terminal lymphocytes) cells can be assessed more precisely [71, 335], depending on the presence or absence of proteins: CD45RA (expression occurs in TN cells), CCR7 (downregulated after interaction with antigen), CD44 (present on the cell surface after interaction with antigen), CD62L (absent on the surface of antigen-experienced T cells) [71, 335]. Each of these markers is an important molecule for T cell maturation and is essential for transition between populations, so two recent publications have used antibodies designed to classify cells based on the presence or absence of each molecule: TN (CD45RA + CD62L +), TCM (CD45RA-CD62L +), TEM (CD45RA-CD62L-), and TEMRA (CD45RA + CD62L-) [336, 337]. Among older dogs, CD4 + and CD8 + cells showed increased expression of TNF-α and IFN-γ in response to mitogen [337]. Older dogs showed reduced TN CD4 + and CD8 + and increased TCM and TEM CD4 + and CD8 + cells compared with young dogs. In contrast, TEMRA CD8 + cells were detected more frequently in older dogs compared to young dogs [337]. An age-related decrease in CD45RA + has also been reported in other studies in both CD4 + and CD8 + populations [73, 328]. In contrast, an increased proportion of CD62L + /CD44 + /CD8 + T cells has been shown in older dogs [338]. This evidence suggests an age-related depletion of naïve CD4 + and CD8 + cells as a result of antigen exposure and the adoption of a memory cell phenotype. Losses in TN, on the other hand, are not replenished quickly enough due to thymic involution. Interestingly, a different profile of changes in the proportion of the T cell population in dogs was obtained by researchers comparing healthy dogs with sick dogs (dogs of similar age). Dermatological inflammation induced an increase in TN CD8 + and a decrease in TEMRA CD8 + [301], confirming the different nature of inflammation induced by aging and that due to disease. The decrease with age in the proliferative capacity of lymphocytes may be the result of decreased IL-2 production with age [72] and decreased IL-2 receptor expression on the lymphocyte surface with age [73], which prevents IL-2-mediated induction of lymphocyte proliferation [339, 340]. In addition, other smaller subsets of lymphocytes (which may differ in their potential to produce cytokines) have been identified that change with age, such as CD4 + and CD8 + CD44 T cells (with different CD44 concentrations), CD4 + CD28 cells (with different CD28 concentrations), and CD5 + T cells, but their association with aging has not yet been well documented. There are canonical differences between CD4 + and CD8 + T cells in terms of the key immunological functions they perform. In the context of severe MMVD (congestive heart failure (CHF) patients), a decrease in CD4 + and CD8 + T cells and a decrease in the CD4 + /CD8 + ratio were similarly detected [341]. The CHF + and CHF- groups were of similar geriatric (11.2 and 10.0 years, respectively) age. Similarly, German Shepherds with mitral valve regurgitation showed a decrease in CD4 + and an increase in CD8 + [342]. A study dedicated to MMVD at earlier stages (B2, C, D ACVIM) showed an increase in CD8 + lymphocytes with disease severity and age dogs, and a decrease in CD4 + lymphocytes with disease severity and age dogs [343]. Additionally, there was a statistically significant difference in age between healthy and diseased dogs [343], which was probably the reason for the lower CD4 + /CD8 + ratio in CHF patients compared to controls [343]. However, in another study, CD4 + FoxP3 + , regulatory T cells (Treg), also undergo decreased abundance with age and also with disease severity in populations of similar age [109]. Moreover, the number of CD8 + was positively associated with echocardiographic indices (left atrial size, mitral inflow characteristics), and CD4 + correlated with them [343]. Furthermore, CD4 + and CD8 + levels are predictive of survival in dogs with MMVD, where cytotoxic lymphocytes correlate negatively with survival, and CD4 + correlates positively with survival [32]. Other changes that lymphocytes undergo include an increase in neutrophil-to-lymphocyte ratio (NLR), and monocyte-to-lymphocyte ratio (MLR) in stage C and D dogs with MMVD [344, 345]. Interestingly, the activity of CD4 + cells in the most severe conditions appears to increase compared to their peers without MMVD [109]. This confirms the relevance of the aging changes that occur in the immune system and may influence the predisposition to assess the health status of an animal with MMVD.

CD8 + cells are Tc lymphocytes that are involved in cell destruction, including tumor cells and SnCs (which are similar in many ways) [346], and recognize major tissue compatibility system (MHC) class I antigens. CD4 + T cells are helper (Th) cells and are much more active in the secretion of inflammatory cytokines. They are divided in dogs into smaller sets: Th1, Th2, and Th17 [71]. In addition, they are cells that recognize antigens bound to MHC class II proteins [71]. Th1 cells, responsible for generating type 1 immune responses, stimulate macrophages and dendritic cells to mount a phagocytic response and direct them to inflammatory sites [71, 347]. Th2, on the other hand, is mainly involved in type 2 immune responses, promotes antiparasitic protection, and regulates wound repair and regeneration pathways [348]. An age-related decrease in MHC-II/CD45RA expression by lymphocytes has been noted [349], and an age-related trend of decreased lymphocyte cytotoxicity [326, 327]. Data have also been obtained indicating a depletion of the lymphocyte TCR receptor repertoire [321, 337], the receptor for specific antigens, and an age-correlated decrease also in CD3 + T cells, the signal transducing molecule of the TCR, leading to T cell activation [332]. In contrast, in the respiratory system, a site of accumulation of immune bodies with numerous clusters of lymphoid tissue (tonsils), a decrease in the number of dendritic cells has been noted in older dogs [350, 351]. There is also emerging evidence of a decrease in the phagocytic capacity of cells induced by, for example, Th and impaired function of the complement system [72, 352]. SnCs secrete SASP, which induces an immune response. In older dogs, this process is impaired, as shown by stimulation by LPS [78]. Thus, changes in phagocytic function and cytotoxicity may contribute to impaired clearance of SnCs in specific organs. However, it is unclear how relevant this process is following a recent study where no significant accumulation of SnCs in the interstitium of the testes was detected in dogs [150]. On the other hand, a significant accumulation of aging cells was obtained in the pulmonary vascular layer in a canine model of pulmonary hypertension, together with an increase in β-galactosidase-positive endothelial progenitor cells [353]. In the aforementioned study, arterial hypertension was induced, but pulmonary venous hypertension, which is also based on endothelial dysfunction, is one of the consequences of developing MMVD. Cardiomyocytes in the aging process in dogs and humans slowly lose efficiency in realizing action potentials [354]; in turn, fibroblasts generate excess ECM as a result of senile transformation and promote the development of diastolic dysfunction [355, 356], and as previously described, qVICs I VEC cells also adopt an active myofibroblast phenotype with concomitant senescence features [33, 117].

Macrophages

Franceschi’s theory of inflammation and network aging devoted a special place to macrophages [29]. Macrophage aging follows a pattern characteristic of other cell types, as evidenced by their impaired phagocytic function and excessive production of inflammatory cytokines [27, 357, 358]. The association of macrophages with aging in dogs still remains elusive. In contrast, in the context of canine MMVD, macrophages have been noted to accumulate at the base of the mitral valve leaflet in increased numbers at different stages of MMVD [126]. Similarly, in humans, macrophages play crucial roles in the development of atherosclerosis. They accumulate in the inflamed vessel wall and release MMPs to degrade the extracellular matrix [359]. In myxomatous lesions in humans and mice, the accumulation of macrophages (CD45 +) was also detected in the subendocardial region, but also in the interstitium of the valves [360]. MMP activity in macrophage localization was elevated [360]. In contrast, mitral valve leaflets with myxomatous degeneration and increased numbers of monocytes and macrophages (CD14 + , CD64 + , CD68 + , CD163 +) were detected in Marfan syndrome pigs [56].

Epigenetic alterations

The knowledge regarding the pathophysiology of MMVD remains elusive since there is a limited amount of research devoted to epigenetic changes, although much progress has recently been made in integrating changes in mi-RNA profiles during MMVD [361]. Some of these find their counterpart in mammalian aging processes (Table 2). An important component of epigenetics, and at the same time of aging, is DNA methylation [362, 363]. Different methylation profiles have been linked to age-related diseases, particularly cardiovascular disease [364]. Similarly, DNA methylation has been used in dogs to identify biological age [338, 365, 366]. However, there is currently a lack of studies dedicated to these processes in the pathophysiology of MMVD. A third, equally important component of epigenetic regulation is histone modification [367]. Since recent reviews, no studies have been made to explain their involvement in aging or in the development of MMVD in aged dogs [134, 135].

Table 2.

Overview on changes in selected mi-RNAs in MMVD

Up-regulated miRNAs related with MMVD	Down-regulated miRNAs related with MMVD
miR-9 [368]	miR-599 [368]
miR-495 [368]	miR-30d [240]
miR-181c [368]	miR-20a [240]
let-7b [280]	let-7c [240]
miR-98 [280]	miR-17 [240]
miR-103 [280]	miR-375 [369]
let-7c [280]	miR-30b-5p [370, 371]
miR-130b [369]	miR-30c [372]
miR-133 [372]	miR-145 [253]
miR-1 [372]	miR-30b [373]
miR-let-7e [372]	miR-423 [372]
miR-125 [372]	miR-128 [372]
	miR-142 [372]
	miR-425 [369]
	miR-30d [369]
	miR-30c [369]
	miR151 [369]
	let-7b [369]
	miR-19b [369]
	let-7 g [369]
	miR-302d [280]
	miR-380 [280]
	miR-874 [280]
	miR-582 [280]
	miR-490 [280]
	miR-329b [280]
	miR-487b [280]

Summary and future directions

The aging of valvular interstitial cells (VICs) appears to be central to unraveling the pathophysiology of MMVD. VICs acquire an aging cell phenotype (aVICs) through canonical cellular senescence processes, as evidenced by studies of DNA damage response pathways. Moreover, like SnCs, aVICs are resistant to apoptosis and exhibit high secretory potential that leads to ECM disruption—in particular, we highlight the role of TGF-β. Several studies have shown that there is a local inflammatory state in mitral valve tissue induced by inflammaging-specific interleukins (IL-6, IL-8, TNF-α), which may contribute to the development or progression of MMVD. The involvement of immune cells in the formation and progression of MMVD in dogs is not as clear as in humans. The detection of macrophage accumulation at the base of the mitral valve is not sufficient evidence, especially in light of the data on inflammation of the inside of the leaflets in human MMVD. The correlation of the TCD4 + population with increased survival of diseased dogs prompts the use of substances with a slowing effect on aging and encourages the use of drugs to maintain the function of this system. Resveratrol, with its effects on main pathways (TGF-β/Smad signaling) and its effects on inflammation and oxidative stress, seems a desirable option. On the other hand, the available gerostatic, rapamycin, reverses the phenotype of aging cells and potentially reduces the deleterious secretory activity of SnC. Pharmacological studies should clarify the appropriate timing for introducing similar drugs and assess their efficacy in dogs and humans. Potential therapeutic targets as preventive solutions or inhibitors of disease progression may be valuable alternatives to treatment with interventional cardiology or cardiac surgery, as these are not yet widely available in veterinary medicine. The absence of a severe fibrosis phase, which distinguishes it from humans, positions the dog as a very good model for the management of this disease in humans in the early stages, as well as in people with a particular predisposition, such as cases of Marfan syndrome. However, the difficulty is in objectively comparing the cellular processes that control disease development in both species. Therefore, there is uncertainty about the efficacy of possible therapies.

Further evidence will probably emerge to support the current findings regarding the epigenetic regulation of VIC phenotypic changes and, in particular, resistance to apoptosis. What triggers the induction of VIC senescence or which type of aging is crucial remains a mystery. Directions for further research, where we see opportunities, should specifically address the epigenetic regulation of the cellular transition of qVICs to aVICs, which would help determine the cause of the differences in lesion appearance in humans and dogs and help identify new therapeutic targets.

Author contribution

All authors have contributed equally.

Funding

A IDUB Mobility Grant was received.

Declarations

Conflict of interest

The authors declare no competing interests.