Endothelial cell sphingosine 1-phosphate receptor 1 restrains VE-cadherin cleavage and attenuates experimental inflammatory arthritis.

In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.

Cardiac Pericytes Acquire a Fibrogenic Phenotype and Contribute to Vascular Maturation After Myocardial Infarction.

BACKGROUND: Pericytes have been implicated in tissue repair, remodeling, and fibrosis. Although the mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. METHODS: We used NG2Dsred;PDGFRαEGFP pericyte:fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in myocardial infarction. The transcriptomic profile of pericyte-derived cells was studied using polymerase chain reaction arrays and single-cell RNA sequencing. The role of transforming growth factor-ß (TGF-ß) signaling in regulation of pericyte phenotype was investigated in vivo using pericyte-specific TGF-ß receptor 2 knockout mice and in vitro using cultured human placental pericytes. RESULTS: In normal hearts, neuron/glial antigen 2 (NG2) and platelet-derived growth factor receptor α (PDGFRα) identified distinct nonoverlapping populations of pericytes and fibroblasts, respectively. After infarction, a population of cells expressing both pericyte and fibroblast markers emerged. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes exhibited transient expression of fibroblast markers. Pericyte-derived cells accounted for ~4% of PDGFRα+ infarct fibroblasts during the proliferative phase of repair. Pericyte-derived fibroblasts were overactive, expressing higher levels of extracellular matrix genes, integrins, matricellular proteins, and growth factors, when compared with fibroblasts from other cellular sources. Another subset of pericytes contributed to infarct angiogenesis by forming a mural cell coat, stabilizing infarct neovessels. Single-cell RNA sequencing showed that NG2 lineage cells diversify after infarction and exhibit increased expression of matrix genes, and a cluster with high expression of fibroblast identity markers emerges. Trajectory analysis suggested that diversification of infarct pericytes may be driven by proliferating cells. In vitro and in vivo studies identified TGF-ß as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific TGF-ß receptor 2 disruption had no significant effects on infarct myofibroblast infiltration and collagen deposition. Pericyte-specific TGF-ß signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels and protecting from dilative remodeling. CONCLUSIONS: In the healing infarct, cardiac pericytes upregulate expression of fibrosis-associated genes, exhibiting matrix-synthetic and matrix-remodeling profiles. A fraction of infarct pericytes exhibits expression of fibroblast identity markers. Pericyte-specific TGF-ß signaling plays a central role in maturation of the infarct vasculature and protects from adverse dilative remodeling, but it does not modulate fibrotic remodeling.

Diabetes Induces Cardiac Fibroblast Activation, Promoting a Matrix-Preserving Nonmyofibroblast Phenotype, Without Stimulating Pericyte to Fibroblast Conversion.

Background Interstitial and perivascular fibrosis may contribute to diabetes-associated heart failure. Pericytes can convert to fibroblasts under conditions of stress and have been implicated in the pathogenesis of fibrotic diseases. We hypothesized that in diabetic hearts, pericytes may convert to fibroblasts, contributing to fibrosis and to the development of diastolic dysfunction. Methods and Results Using pericyte:fibroblast dual reporter (NG2Dsred [neuron-glial antigen 2 red fluorescent protein variant]; PDGFRαEGFP [platelet-derived growth factor receptor alpha enhanced green fluorescent protein]) mice in a type 2 diabetic db/db background, we found that diabetes does not significantly affect pericyte density but reduces the myocardial pericyte:fibroblast ratio. Lineage tracing using the inducible NG2CreER driver, along with reliable labeling of fibroblasts with the PDGFRα reporter system, showed no significant pericyte to fibroblast conversion in lean and db/db hearts. In addition, db/db mouse cardiac fibroblasts did not undergo myofibroblast conversion and had no significant induction of structural collagens but exhibited a matrix-preserving phenotype, associated with increased expression of antiproteases, matricellular genes, matrix cross-linking enzymes, and the fibrogenic transcription factor cMyc. In contrast, db/db mouse cardiac pericytes had increased expression of Timp3, without any changes in expression of other fibrosis-associated genes. The matrix-preserving phenotype of diabetic fibroblasts was associated with induction of genes encoding oxidative (Ptgs2/cycloxygenase-2, and Fmo2) and antioxidant proteins (Hmox1, Sod1). In vitro, high glucose partially recapitulated the in vivo changes in diabetic fibroblasts. Conclusions Diabetic fibrosis is not mediated through pericyte to fibroblast conversion but involves acquisition of a matrix-preserving fibroblast program, which is independent of myofibroblast conversion and is only partially explained by the effects of the hyperglycemic environment.

Identification of macrophages in normal and injured mouse tissues using reporter lines and antibodies.

Reliable tools for macrophage identification in mouse tissues are critical for studies investigating inflammatory and reparative responses. Transgenic reporter mice and anti-macrophage antibodies have been used as "specific pan-macrophage" markers in many studies; however, organ-specific patterns of expression and non-specific labeling of other cell types, such as fibroblasts, may limit their usefulness. Our study provides a systematic comparison of macrophage labeling patterns in normal and injured mouse tissues, using the CX3CR1 and CSF1R macrophage reporter lines and anti-macrophage antibodies. Moreover, we tested the specificity of macrophage antibodies using the fibroblast-specific PDGFR[Formula: see text] reporter line. Mouse macrophages exhibit organ-specific differences in expression of macrophage markers. Hepatic macrophages are labeled for CSF1R, Mac2 and F4/80, but lack CX3CR1 expression, whereas in the lung, the CSF1R+/Mac2+/Mac3+ macrophage population is not labeled with F4/80. In the splenic red pulp, subpopulations of CSF1R+/F4/80+/Mac3+cells were labeled with Mac2, CX3CR1 and lysozyme M. In the kidney, Mac2, Mac3 and lysozyme M labeled a fraction of the CSF1R+ and CX3CR1+ macrophages, but also stained tubular epithelial cells. In normal hearts, the majority of CSF1R+ and CX3CR1+ cells were not detected with anti-macrophage antibodies. Myocardial infarction was associated with marked expansion of the CSF1R+ and CX3CR1+ populations that peaked during the proliferative phase of cardiac repair, and also expressed Mac2, Mac3 and lysozyme M. In normal mouse tissues, a small fraction of cells labeled with anti-macrophage antibodies were identified as PDGFR[Formula: see text]+ fibroblasts, using a reporter system. The population of PDGFR[Formula: see text]+ cells expressing macrophage markers expanded following injury, likely reflecting emergence of cellular phenotypes with both fibroblast and macrophage characteristics. In conclusion, mouse macrophages exhibit remarkable heterogeneity. Selection of the most appropriate markers for identification of macrophages in mouse tissues is dependent on the organ and the pathologic condition studied.

Validation of Specific and Reliable Genetic Tools to Identify, Label, and Target Cardiac Pericytes in Mice.

Background In the myocardium, pericytes are often confused with other interstitial cell types, such as fibroblasts. The lack of well-characterized and specific tools for identification, lineage tracing, and conditional targeting of myocardial pericytes has hampered studies on their role in heart disease. In the current study, we characterize and validate specific and reliable strategies for labeling and targeting of cardiac pericytes. Methods and Results Using the neuron-glial antigen 2 (NG2)DsRed reporter line, we identified a large population of NG2+ periendothelial cells in mouse atria, ventricles, and valves. To examine possible overlap of NG2+ mural cells with fibroblasts, we generated NG2DsRed; platelet-derived growth factor receptor (PDGFR) αEGFP pericyte/fibroblast dual reporter mice. Myocardial NG2+ pericytes and PDGFRα+ fibroblasts were identified as nonoverlapping cellular populations with distinct transcriptional signatures. PDGFRα+ fibroblasts expressed high levels of fibrillar collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and genes encoding matricellular proteins, whereas NG2+ pericytes expressed high levels of Pdgfrb, Adamts1, and Vtn. To validate the specificity of pericyte Cre drivers, we crossed these lines with PDGFRαEGFP fibroblast reporter mice. The constitutive NG2Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRßCreER line labeled not only mural cells but also the majority of cardiac and aortic fibroblasts. Conclusions Fibroblasts and pericytes are topographically and transcriptomically distinct populations of cardiac interstitial cells. The inducible NG2CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRßCreER line lacks specificity.

Smad7 effects on TGF-ß and ErbB2 restrain myofibroblast activation and protect from postinfarction heart failure.

Repair of the infarcted heart requires TGF-ß/Smad3 signaling in cardiac myofibroblasts. However, TGF-ß-driven myofibroblast activation needs to be tightly regulated in order to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of the inhibitory Smad, Smad7, may restrain infarct myofibroblast activation, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of nonreperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA+ infarct myofibroblasts, but not in α-SMA-PDGFRα+ fibroblasts. Myofibroblast-specific Smad7 loss increased heart failure-related mortality, worsened dysfunction, and accentuated fibrosis in the infarct border zone and in the papillary muscles. Smad7 attenuated myofibroblast activation and reduced synthesis of structural and matricellular extracellular matrix proteins. Smad7 effects on TGF-ß cascades involved deactivation of Smad2/3 and non-Smad pathways, without any effects on TGF-ß receptor activity. Unbiased transcriptomic and proteomic analysis identified receptor tyrosine kinase signaling as a major target of Smad7. Smad7 interacted with ErbB2 in a TGF-ß-independent manner and restrained ErbB1/ErbB2 activation, suppressing fibroblast expression of fibrogenic proteases, integrins, and CD44. Smad7 induction in myofibroblasts serves as an endogenous TGF-ß-induced negative feedback mechanism that inhibits postinfarction fibrosis by restraining Smad-dependent and Smad-independent TGF-ß responses, and by suppressing TGF-ß-independent fibrogenic actions of ErbB2.

Collagen denaturation in the infarcted myocardium involves temporally distinct effects of MT1-MMP-dependent proteolysis and mechanical tension.

Tissue injury results in profound alterations in the collagen network, associated with unfolding of the collagen triple helix, proteolytic degradation and generation of fragments. In the infarcted myocardium, changes in the collagen network are critically involved in the pathogenesis of left ventricular rupture, adverse remodeling and chronic dysfunction. We hypothesized that myocardial infarction is associated with temporally and spatially restricted patterns of collagen denaturation that may reflect distinct molecular mechanisms of collagen unfolding. We used a mouse model of non-reperfused myocardial infarction, and in vitro assays in fibroblast-populated collagen lattices. In healing infarcts, labeling with collagen hybridizing peptide (CHP) revealed two distinct patterns of collagen denaturation. During the inflammatory and proliferative phases of infarct healing, collagen denaturation was pericellular, localized in close proximity to macrophages and myofibroblasts. qPCR array analysis of genes associated with matrix remodeling showed that Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) is markedly upregulated in infarct macrophages and fibroblasts, suggesting its involvement in pericellular collagen denaturation. In vitro, MT1-MMP-mediated pericellular collagen denaturation is involved in cardiac fibroblast migration. The effects of MT1-MMP on collagen denaturation and fibroblast migration involve the catalytic site, and require hemopexin domain-mediated actions. In contrast, during the maturation phase of infarct healing, extensive collagen denaturation was noted in the hypocellular infarct, in the infarct border zone and in the mitral valve annulus, in the absence of MT1-MMP. In vitro, mechanical tension in attached collagen lattices was sufficient to induce peripheral collagen denaturation. Our study suggests that in healing infarcts, early pericellular collagen denaturation may be important for migration of macrophages and reparative myofibroblasts in the infarct. Extensive denaturation of collagen fibers is noted in mature scars, likely reflecting mechanical tension. Chronic collagen denaturation may increase susceptibility of the matrix to proteolysis, thus contributing to progressive cardiac dilation and post-infarction heart failure.

The role of Smad2 and Smad3 in regulating homeostatic functions of fibroblasts in vitro and in adult mice.

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-ß-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF.

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

TGF-ßs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-ß1, -ß2 and -ß3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Pericytes in the infarcted heart.

The adult mammalian heart lacks regenerative capacity and heals through activation of an inflammatory cascade that leads to the formation of a collagen-based scar. Although scar formation is important to preserve the structural integrity of the ventricle, unrestrained inflammation and excessive fibrosis have been implicated in the pathogenesis of adverse post-infarction remodeling and heart failure. Interstitial cells play a crucial role in the regulation of cardiac repair. Although recent studies have explored the role of fibroblasts and immune cells, the cardiac pericytes have been largely ignored by investigators interested in myocardial biology. This review manuscript discusses the role of pericytes in the regulation of inflammation, fibrosis and angiogenesis following myocardial infarction. During the inflammatory phase of infarct healing, pericytes may regulate microvascular permeability and may play an important role in leukocyte trafficking. Moreover, pericyte activation through Toll-like receptor-mediated pathways may stimulate cytokine and chemokine synthesis. During the proliferative phase, pericytes may be involved in angiogenesis and fibrosis. To what extent pericyte to fibroblast conversion and pericyte-mediated growth factor synthesis contribute to the myocardial fibrotic response remains unknown. During the maturation phase of infarct healing, coating of infarct neovessels with pericytes plays an important role in scar stabilization. Implementation of therapeutic approaches targeting pericytes in the infarcted and remodeling heart remains challenging, due to the lack of systematic characterization of myocardial pericytes, their phenotypic heterogeneity and the limited knowledge on their functional role.

Characterization of a mouse model of obesity-related fibrotic cardiomyopathy that recapitulates features of human heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is caused, or exacerbated by, a wide range of extracardiac conditions. Diabetes, obesity, and metabolic dysfunction are associated with a unique HFpEF phenotype, characterized by inflammation, cardiac fibrosis, and microvascular dysfunction. Development of new therapies for HFpEF is hampered by the absence of reliable animal models. The leptin-resistant db/ db mouse has been extensively studied as a model of diabetes-associated cardiomyopathy; however, data on the functional and morphological alterations in db/ db hearts are conflicting. In the present study, we report a systematic characterization of the cardiac phenotype in db/ db mice, focusing on the time course of functional and histopathological alterations and on the identification of sex-specific cellular events. Although both male and female db/ db mice developed severe obesity, increased adiposity, and hyperglycemia, female mice had more impressive weight gain and exhibited a modest but significant increase in blood pressure. db/ db mice had hypertrophic ventricular remodeling and diastolic dysfunction with preserved ejection fraction; the increase in left ventricular mass was accentuated in female mice. Histological analysis showed that both male and female db/ db mice had cardiomyocyte hypertrophy and interstitial fibrosis, associated with marked thickening of the perimysial collagen, and expansion of the periarteriolar collagen network, in the absence of replacement fibrosis. In vivo and in vitro experiments showed that fibrotic changes in db/ db hearts were associated with increased collagen synthesis by cardiac fibroblasts, in the absence of periostin, α-smooth muscle actin, or fibroblast activation protein overexpression. Male db/ db mice exhibited microvascular rarefaction. In conclusion, the db/ db mouse model recapitulates functional and histological features of human HFpEF associated with metabolic dysfunction. Development of fibrosis in db/ db hearts, in the absence of myofibroblast conversion, suggests that metabolic dysfunction may activate an alternative profibrotic pathway associated with accentuated extracellular matrix protein synthesis. NEW & NOTEWORTHY We provide a systematic analysis of the sex-specific functional and structural myocardial alterations in db/ db mice. Obese diabetic C57BL6J db/ db mice exhibit diastolic dysfunction with preserved ejection fraction, associated with cardiomyocyte hypertrophy, interstitial/perivascular fibrosis, and microvascular rarefaction, thus recapitulating aspects of human obesity-related heart failure with preserved ejection fraction. Myocardial fibrosis in db/ db mice is associated with a matrix-producing fibroblast phenotype, in the absence of myofibroblast conversion, suggesting an alternative mechanism of activation.

Left atrial remodeling, hypertrophy, and fibrosis in mouse models of heart failure.

Left ventricular dysfunction increases left atrial pressures and causes atrial remodeling. In human subjects, increased left atrial size is a powerful predictor of mortality and adverse events in a broad range of cardiac pathologic conditions. Moreover, structural remodeling of the atrium plays an important role in the pathogenesis of atrial tachyarrhythmias. Despite the potential value of the atrium in assessment of functional endpoints in myocardial disease, atrial pathologic alterations in mouse models of left ventricular disease have not been systematically investigated. Our study describes the geometric, morphologic, and structural changes in experimental mouse models of cardiac pressure overload (induced through transverse aortic constriction), myocardial infarction, and diabetes. Morphometric and histological analysis showed that pressure overload was associated with left atrial dilation, increased left atrial mass, loss of myofibrillar content in a subset of atrial cardiomyocytes, atrial cardiomyocyte hypertrophy, and atrial fibrosis. In mice undergoing nonreperfused myocardial infarction protocols, marked left ventricular systolic dysfunction was associated with left atrial enlargement, atrial cardiomyocyte hypertrophy, and atrial fibrosis. Both infarcted animals and pressure overloaded mice exhibited attenuation and perturbed localization of atrial connexin-43 immunoreactivity, suggesting gap junctional remodeling. In the absence of injury, obese diabetic db/db mice had diastolic dysfunction associated with atrial dilation, atrial cardiomyocyte hypertrophy, and mild atrial fibrosis. Considering the challenges in assessment of clinically relevant functional endpoints in mouse models of heart disease, study of atrial geometry and morphology may serve as an important new tool for evaluation of ventricular function.