Mark C. Blaser

Animal Models of Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD), once thought to be a degenerative disease, is now recognized to be an active pathobiological process, with chronic inflammation emerging as a predominant, and possibly driving, factor. However, many details of the pathobiological mechanisms of CAVD remain to be described, and new approaches to treat CAVD need to be identified. Animal models are emerging as vital tools to this end, facilitated by the advent of new models and improved understanding of the utility of existing models. In this paper, we summarize and critically appraise current small and large animal models of CAVD, discuss the utility of animal models for priority CAVD research areas, and provide recommendations for future animal model studies of CAVD.

Inhibition of Pathological Differentiation of Valvular Interstitial Cells by C-Type Natriuretic Peptide

Objective—Calcific aortic valve disease is associated with the differentiation of valvular interstitial cells (VICs) to myofibroblast and osteoblast-like cells, particularly in the fibrosa layer of the valve. Previous studies suggested that C-type natriuretic peptide (CNP) protects against calcific aortic valve disease to maintain homeostasis. We aimed to determine whether CNP inhibits VIC pathological differentiation as a mechanism to explain its protective effects. Methods and Results—CNP expression was prominent in normal porcine aortic valves, particularly on the ventricular side, but reduced in sclerotic valves concomitant with the appearance of pathological VIC phenotypes in the fibrosa. In vitro, CNP inhibited calcified aggregate formation and bone-related transcript and protein expression by VICs grown in osteogenic conditions. Under myofibrogenic culture conditions, CNP reduced &agr;-smooth muscle actin expression and cell-mediated gel contraction, indicating inhibition of myofibroblast differentiation. Similar to CNP, simvastatin inhibited VIC osteoblast and myofibroblast differentiation in vitro. Strikingly, simvastatin upregulated CNP expression in VICs cultured under myofibrogenic conditions, and small interfering RNA knockdown of natriuretic peptide receptor-b (a CNP receptor) significantly reduced the antifibrotic effect of simvastatin, suggesting that it acts in part via CNP/NPR-B autocrine/paracrine signaling. Conclusion—CNP inhibits myofibroblast and osteoblast differentiation of VICs and is responsible in part for inhibition of VIC myofibroblast differentiation by statins, suggesting novel mechanisms to explain the protective effect of CNP and the pleiotropic effects of statins in the aortic valve.

Morphological and Functional Evidence for the Contribution of the Pudendal Artery in Aging-Induced Erectile Dysfunction

INTRODUCTION Aging increases the risk of both erectile dysfunction (ED) and cardiovascular disease. These conditions have similar etiologies and commonly coexist. One unifying concept is the role of arterial insufficiency which is a primary factor in the onset of age-related ED. AIM Based on the novel finding that the pudendal arteries contribute 70% of the total penile vascular resistance, our objective was to morphometrically and functionally characterize this vessel in young and old normotensive rats. METHODS Erectile function was monitored in 15- and 77-week Sprague-Dawley rats using the apomorphine bioassay (80mg/kg, s.c.). Anesthetized animals were perfusion-fixed, aortic, renal, and internal pudendal arteries were excised, embedded, sectioned, stained, and morphometrically assessed using light microscopy. Hearts were excised, separated, and weighed prior to perfusion. Contractile and relaxation responses to acetylcholine (ACh) and phenylephrine (PE) were assessed by wire myograph. MAIN OUTCOME MEASURES Erectile function, morphological measurements, concentration response curves to ACh and PE. RESULTS With age, there were marked decreases in erectile responses compared to younger rats (2.8±0.87 vs. 0.3±0.58). The pudendal arteries had a relatively small lumen (303±13.8µm) and a thick medial layer (47±2.2µm). In aged pudendal arteries, the lumen diameter did not change, and yet the medial layer, cross sectional area, and extracellular matrix were markedly increased. In contrast, the lumen diameter and wall thickness of the aorta and renal arteries in aged rats increased proportionally. An increase in small, round, smooth muscle cells was seen in aged pudendal arteries. Functionally, there were no differences in contractile responses to PE; however, ACh-induced relaxation decreased with age. CONCLUSIONS In aged rats, erectile function was severely diminished when pudendal arteries had undergone marked phenotypic changes. Specifically, there was endothelial dysfunction and pathological remodeling of this vessel with age, characterized by medial thickening, impaired vasodilation and significantly reduced capacity for penile blood flow.

Biomechanical conditioning of tissue engineered heart valves: Too much of a good thing?

Surgical replacement of dysfunctional valves is the primary option for the treatment of valvular disease and congenital defects. Existing mechanical and bioprosthetic replacement valves are far from ideal, requiring concomitant anticoagulation therapy or having limited durability, thus necessitating further surgical intervention. Heart valve tissue engineering (HVTE) is a promising alternative to existing replacement options, with the potential to synthesize mechanically robust tissue capable of growth, repair, and remodeling. The clinical realization of a bioengineered valve relies on the appropriate combination of cells, biomaterials, and/or bioreactor conditioning. Biomechanical conditioning of valves in vitro promotes differentiation of progenitor cells to tissue-synthesizing myofibroblasts and prepares the construct to withstand the complex hemodynamic environment of the native valve. While this is a crucial step in most HVTE strategies, it also may contribute to fibrosis, the primary limitation of engineered valves, through sustained myofibrogenesis. In this review, we examine the progress of HVTE and the role of mechanical conditioning in the synthesis of mechanically robust tissue, and suggest approaches to achieve myofibroblast quiescence and prevent fibrosis.

Impact of Hypertension, Aging, and Antihypertensive Treatment on the Morphology of the Pudendal Artery

INTRODUCTION Aging and hypertension increase the risk of erectile dysfunction (ED) and cardiovascular disease. Arterial insufficiency is likely a primary factor in hypertension-related ED. Given the dominance of internal pudendal arteries in controlling penile vascular resistance, pathological changes in this vessel would be critical for inducing ED in aged hypertensives. AIM We assessed the age-related impact of hypertension and its treatment on erectile function and pudendal artery structure in young and old spontaneously hypertensive rats (SHRs). METHODS Erectile responses were monitored in 15- and 77-week-old SHR and Wistar Kyoto (WKY) rats using apomorphine (80 mg/kg). At sacrifice, the vasculature was perfusion-fixed and aorta, renal, mesenteric, and internal pudendal arteries assessed morphometrically using light and electron microscopy. A separate group of 15-week SHR were treated with enalapril and hydrochlorothiazide (30 mg/kg/day, 2 weeks) followed by 2 weeks off treatment, after which the same vessels were assessed morphometrically. Arterial pressures were determined using radiotelemetry. MAIN OUTCOMES MEASURED Erectile function, vessel morphology (lumen diameter, wall thickness, cross-sectional area, extracellular matrix [ECM]) and arterial pressure. RESULTS Erectile responses were similar in young SHR and WKY (1.7 ± 0.80 vs. 1.4 ± 0.85) but declined significantly in aged SHR (0.3 ± 0.49). Vascular aging in SHR was associated with striking pudendal remodeling, characterized by marked neointimal proliferation and disruptions of the internal elastic lamina. This remodeling involved thickening of the medial layer (35 ± 6.0 µm vs. 81 ± 3.5 µm, P < 0.01), decreased lumen diameter (282 ± 6.3 µm vs. 250 ± 12.4 µm, P < 0.05) and increased ECM (10 ± 2.0 µm² vs. 26 ± 10.6 µm², P < 0.001). In old pudendals, there were significantly more round synthetic smooth muscle cells bordering the intima and in the neointima. Antihypertensive treatment decreased the wall:lumen ratio in young SHR pudendal arteries (-17%). CONCLUSIONS Vascular aging in SHR with ED involved distinctive pathogenic remodeling in the internal pudendal artery. In young SHR, brief antihypertensive therapy was able to regress this abnormal morphology.

Characterization of the Vasculature Supplying the Genital Tissues in Female Rats

INTRODUCTION The internal pudendal arteries are the key resistance vessels controlling the peripheral circulatory component of sexual responses in both male and females. Previous studies in the male rat demonstrated that this vessel has markedly heightened susceptibility to vascular damage compared with other vessels in the body. Evidence suggests that the female may also be susceptible to vascular pathologies contributing to sexual dysfunction. AIM The aim of this study is to characterize the anatomical, morphological, and functional properties of the pudendal artery in female rats. METHODS The pelvic arteries in young Sprague-Dawley female rats were dissected to generate a composite representation of the vascular gross anatomy. Morphometry was performed on perfusion-fixed pudendal arteries whereas others were mounted in a wire myograph to assess responses to vasoactive drugs. These measures were contrasted with a previous study examining male rats. MAIN OUTCOME MEASURES Outcome measures used are gross anatomy, lumen diameter, wall thickness, cross-sectional area, and contractile responses in the internal pudendal artery. RESULTS The gross anatomy of the pudendal artery in female rats appears to parallel that found in male rats, acting as the primary feeder vessel of the clitoral, labial, and vaginal tissue. Compared with the male rat, the female pudendal artery has a smaller lumen diameter (169 ± 5.7 vs. 303 ± 13.8 µm), wall thickness (14 ± 0.7 vs. 47 ± 2.2 µm), and cross-sectional area (8 ± 0.4 vs. 52 ± 3.4 × 103 µm(2) ). These structural differences also translate into a decreased contractile capacity of the pudendal arteries from female rats vs. male rats (8.1 ± 2.7 vs. 20 ± 1.4 mN). CONCLUSIONS Although the gross anatomical features of the vasculature tree supplying the genital tissue in male and female rats appear to have similarities, the tissue-specific properties of the vessel itself have a very different structure-function balance. We hypothesize that this discordance likely reflects the very different sex-specific roles of this vessel in regulating blood flow during arousal.

Giving Calcification Its Due: Recognition of a Diverse Disease: A First Attempt to Standardize the Field.

Cardiovascular calcification is a growing burden and a leading predictor of and contributor to acute cardiovascular events. Arterial calcification associates with a 4-fold increase in cardiovascular events, and patients with aortic valve calcification have a 5-year event-free survival rate of only 26%,1,2 worse than that of many cancers. Despite massive healthcare costs and extensive research efforts, effective therapeutic strategies remain elusive. Cardiovascular calcification, often generalized as one disease, is in reality a multifaceted disorder that occurs in diverse milieus as a result of multiple, interacting pathogenic processes. Herein, we explore these variables and propose a set of guidelines for studying cardiovascular calcification. Although vascular and valvular calcification share several risk factors, only ≈25% to 50% of patients with aortic valve calcification also present with significant coronary artery disease,3 suggesting that common risk factors initiate divergent disease processes. ### Vascular Versus Valvular Calcification Differences in prevalence are not unexpected, particularly when these 2 tissues are closely examined. Unlike the collagen and elastin-rich medial layer of the vasculature, the aortic valve has a complex trilayered architecture consisting of collagen, elastin, and proteoglycans. Calcific nodules occur almost exclusively on the stiffer, collagen-rich aortic side of the valve. Calcification takes the form of either hyperphosphatemic medial mineralization or inflammatory-driven intimal calcification, and although these discrete forms may coexist, they tend to be mechanistically distinct. Only 10% to 13% of valves and vasculature exhibit mature bone formation, while the remainder is dystrophic mineralization.4 The resident cell populations responsible for maintaining these tissue structures are also dissimilar: instead of vascular smooth muscle cells, valve interstitial cells populate the aortic valve. Valve interstitial cells are normally a quiescent fibroblastic population that may undergo myofibrogenesis, osteogenesis, and chondrogenesis during disease, thereby adding complexity to valvular calcification. The valve is particularly sensitive to biomechanical stimuli—calcific nodule formation occurs in …

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in MiceNovelty and Significance

Rationale: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. Objective: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. Methods and Results: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein–coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/− and Npr2+/−;Ldlr−/− mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/− fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/− and Npr2+/−;Ldlr−/− mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/− and Npr2+/−;Ldlr−/− mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/− with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/− valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/−;Ldlr−/− mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/− mice with bicuspid aortic valves than those with tricuspid valves. Conclusions: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

Spatiotemporal Multi-omics Mapping Generates a Molecular Atlas of the Aortic Valve and Reveals Networks Driving Disease

Background: No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without invasive valve replacement. The search for therapeutics and early diagnostics is challenging because CAVD presents in multiple pathological stages. Moreover, it occurs in the context of a complex, multi-layered tissue architecture; a rich and abundant extracellular matrix phenotype; and a unique, highly plastic, and multipotent resident cell population. Methods: A total of 25 human stenotic aortic valves obtained from valve replacement surgeries were analyzed by multiple modalities, including transcriptomics and global unlabeled and label-based tandem-mass-tagged proteomics. Segmentation of valves into disease stage–specific samples was guided by near-infrared molecular imaging, and anatomic layer-specificity was facilitated by laser capture microdissection. Side-specific cell cultures were subjected to multiple calcifying stimuli, and their calcification potential and basal/stimulated proteomes were evaluated. Molecular (protein–protein) interaction networks were built, and their central proteins and disease associations were identified. Results: Global transcriptional and protein expression signatures differed between the nondiseased, fibrotic, and calcific stages of CAVD. Anatomic aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein as a specific marker of valvular interstitial cells from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that in vitro, fibrosa-derived valvular interstitial cells demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured valvular interstitial cells, and that valvular interstitial cells exposed to alkaline phosphatase–dependent and alkaline phosphatase–independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Analysis of protein–protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions: A spatially and temporally resolved multi-omics, and network and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a novel means of systematic disease ontology that is broadly applicable to comprehensive omics studies of cardiovascular diseases.

Mechanical and Matrix Regulation of Valvular Fibrosis

The aortic valve lies in, arguably, one of the more complex local mechanobiological environments in the body. The inherent intricacy of this microenvironment results in multiple homeostatic mechanisms, but also a wide variety of putative disease pathways by which valve function can be compromised. Aortic valve disease (AVD) is a cell-mediated pathology whose initial stages are characterized by unchecked matrix dysregulation, leaflet thickening, and widespread fibrosis. The valve itself is composed of multiple cell populations, including endothelial cells that are sensitive to blood flow-induced shear stresses and multipotent mesenchymal progenitors which are influenced by both the mechanical properties and composition of the surrounding extracellular matrix. Dynamic mechanical loading and shear stresses over the cardiac cycle, an irregular three-dimensional shape, and a non-uniform matrix composition further influence these cellular responses. There is also abundant biochemical signaling in the aortic root, with molecular factors either produced by valve cells or transported to the root via blood flow. When these mechanical/biochemical processes become deregulated as a result of insults to their constituent components, resident valvular cells are driven to undergo myofibroblastic differentiation, a program of valvular fibrosis sets in, and valve function is compromised. Valve dysfunction affects the cardiac environment as well, as impaired opening and reductions in orifice area alter myocardial mechanics and often result in hypertrophy and/or fibrosis of the left ventricle. In this chapter, we use the aortic valve as a model tissue to discuss causative mechanisms of cardiovascular fibrosis, including the contributions of mechanotransduction, matrix dysregulation, and biochemical signaling.