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 NG2 DsRed ; platelet‐derived growth factor receptor (PDGFR) α EGFP 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 PDGFRα EGFP fibroblast reporter mice. The constitutive NG2 Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2 CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRβ CreER 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 NG2 CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRβ CreER line lacks specificity.

Modulation of Cardiac Fibrosis in and Beyond Cells

The extracellular matrix (ECM) plays important roles in maintaining physiological structure and functions of various tissues and organs. Cardiac fibrosis is the excess deposition of ECM, including both fibrillar (collagens I and III) and non-fibrillar proteins. Characteristics of fibrosis can vary depending on the pathology, with focal fibrosis occurring following myocardial infarction (MI), and diffuse interstitial and perivascular fibrosis mainly in non-ischemic heart diseases. Compliance of the fibrotic tissue is significantly lower than the normal myocardium, and this can compromise the diastolic, as well as systolic dysfunction. Therefore, strategies to combat cardiac fibrosis have been investigated. Upon injury or inflammation, activated cardiac fibroblasts (myofibroblasts) produce more ECM proteins and cause fibrosis. The activation could be inhibited or the myofibroblasts could be ablated by targeting their specific expressed proteins. Modulation of tissue inhibitors of metalloproteinases (TIMPs) and moderate exercise can also suppress cardiac fibrosis. More recently, sex differences in cardiac fibrosis have come to light with differential fibrotic response in heart diseases as well as in fibroblast functions in vitro. This mini-review discusses recent progress in cardiac fibroblasts, TIMPs, sex differences and exercise in modulation of cardiac fibrosis.

Ubiquitin Ligase Wwp1 Gene Deletion Attenuates Diastolic Dysfunction in Pressure Overload Hypertrophy

Background. Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and accompanied by abnormal extracellular matrix (ECM) accumulation. The E3 ubiquitin ligase WWP1 is a fundamental determinant ECM turnover. We tested the hypothesis that genetic ablation of Wwp1 would alter the progression of LVPO induced HFpEF. Methods/Results. LV echocardiography in mice with global Wwp1 deletion (n=41; Wwp1-/-) was performed at 12 weeks of age (Baseline) and then at 2 and 4 weeks following LVPO (transverse aortic banding) or surgery without LVPO induction. Age-matched wild type mice (Wwp1+/+; n=33) underwent identical protocols. LV EF remained constant and unchanged with LVPO and LV mass increased in both groups but was lower in the Wwp1-/- mice. With LVPO, the E/A ratio, an index of LV filling, was 3.97 + 0.46 in Wwp1+/+ but was 1.73 + 0.19 in the Wwp1-/- group (p<0.05). At the transcriptional level, mRNA for fibrillar collagens (types I and III) decreased by approximately 50% in Wwp1-/- compared to the Wwp1+/+ group at 4 weeks post-LVPO (p<0.05) and was paralleled by a similar difference in LV fibrillar collagen content as measured by histochemistry. Moreover, mRNA levels for determinants favoring ECM accumulation, such as transforming growth factor (TGF) increased with LVPO, but were lower in the Wwp1-/- group. Summary. The absence of Wwp1 reduced the development of LVH and subsequent progression to HFpEF. Modulating the WWP1 pathway could be a therapeutic target to alter the natural history of HFpEF.

Transcriptome and proteome dynamics of cervical remodeling in the mouse during pregnancy

During gestation, the female reproductive tract must maintain pregnancy while concurrently preparing for parturition. Here, we explore the transitions in gene expression and protein turnover (fractional synthesis rates [FSR]) by which the cervix implements a transition from rigid to compliant. Shifts in gene transcription to achieve immune tolerance and alter epithelial cell programs begin in early pregnancy. Subsequently, in mid-to-late pregnancy transcriptional programs emerge that promote structural reorganization of the extracellular matrix (ECM). Stable isotope labeling revealed a striking slowdown of overall FSRs across the proteome on gestation day 6 that reverses in mid-to-late pregnancy. An exception was soluble fibrillar collagens and proteins of collagen assembly, which exhibit high turnover in non-pregnant cervix compared to other tissues and FSRs that continue throughout pregnancy. This finding provides a mechanism to explain how cross-linked collagen is replaced by newly synthesized, less-cross-linked collagens, which allows increased tissue compliance during parturition. The rapid transition requires a reservoir of newly synthesized, less cross-linked collagens, which is assured by the high FSR of soluble collagens in the cervix. These findings suggest a previously unrecognized form of “metabolic flexibility” for ECM in the cervix that underlies rapid transformation in compliance to allow parturition.

Suppression of pancreatic ductal adenocarcinoma growth and metastasis by fibrillar collagens produced selectively by tumor cells

Pancreatic ductal adenocarcinoma (PDAC) has a collagen-rich dense extracellular matrix (ECM) that promotes malignancy of cancer cells and presents a barrier for drug delivery. Data analysis of our published mass spectrometry (MS)-based studies on enriched ECM from samples of progressive PDAC stages reveal that the C-terminal prodomains of fibrillar collagens are partially uncleaved in PDAC ECM, suggesting reduced procollagen C-proteinase activity. We further show that the enzyme responsible for procollagen C-proteinase activity, bone morphogenetic protein1 (BMP1), selectively suppresses tumor growth and metastasis in cells expressing high levels of COL1A1. Although BMP1, as a secreted proteinase, promotes fibrillar collagen deposition from both cancer cells and stromal cells, only cancer-cell-derived procollagen cleavage and deposition suppresses tumor malignancy. These studies reveal a role for cancer-cell-derived fibrillar collagen in selectively restraining tumor growth and suggest stratification of patients based on their tumor epithelial collagen I expression when considering treatments related to perturbation of fibrillar collagens.

Collagen Assembly at the Cell Surface: Dogmas Revisited

With the increased awareness about the importance of the composition, organization, and stiffness of the extracellular matrix (ECM) for tissue homeostasis, there is a renewed need to understand the details of how cells recognize, assemble and remodel the ECM during dynamic tissue reorganization events. Fibronectin (FN) and fibrillar collagens are major proteins in the ECM of interstitial matrices. Whereas FN is abundant in cell culture studies, it is often only transiently expressed in the acute phase of wound healing and tissue regeneration, by contrast fibrillar collagens form a persistent robust scaffold in healing and regenerating tissues. Historically fibrillar collagens in interstitial matrices were seen merely as structural building blocks. Cell anchorage to the collagen matrix was thought to be indirect and occurring via proteins like FN and cell surface-mediated collagen fibrillogenesis was believed to require a FN matrix. The isolation of four collagen-binding integrins have challenged this dogma, and we now know that cells anchor directly to monomeric forms of fibrillar collagens via the α1β1, α2β1, α10β1 and α11β1 integrins. The binding of these integrins to the mature fibrous collagen matrices is more controversial and depends on availability of integrin-binding sites. With increased awareness about the importance of characterizing the total integrin repertoire on cells, including the integrin collagen receptors, the idea of an absolute dependence on FN for cell-mediated collagen fibrillogenesis needs to be re-evaluated. We will summarize data suggesting that collagen-binding integrins in vitro and in vivo are perfectly well suited for nucleating and supporting collagen fibrillogenesis, independent of FN.

Proteomic Analysis of Porcine-Derived Collagen Membrane and Matrix

Collagen membranes and matrices being widely used in guided bone regeneration and soft tissue augmentation have characteristic properties based on their composition. The respective proteomic signatures have not been identified. Here, we performed a high-resolution shotgun proteomic analysis on two porcine collagen-based biomaterials designed for guided bone regeneration and soft tissue augmentation. Three lots each of a porcine-derived collagen membrane and a matrix derived from peritoneum and/or skin were digested and separated by nano-reverse-phase high-performance liquid chromatography. The peptides were subjected to mass spectrometric detection and analysis. A total of 37 proteins identified by two peptides were present in all collagen membranes and matrices, with 11 and 16 proteins being exclusively present in the membrane and matrix, respectively. The common extracellular matrix proteins include fibrillar collagens (COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL5A3, COL11A2), non-fibrillar collagens (COL4A2, COL6A1, COL6A2, COL6A3, COL7A1, COL16A1, COL22A1), and leucine-rich repeat proteoglycans (DCN, LUM, BGN, PRELP, OGN). The structural proteins vimentin, actin-based microfilaments (ACTB), annexins (ANXA1, ANXA5), tubulins (TUBA1B, TUBB), and histones (H2A, H2B, H4) were also identified. Examples of membrane-only proteins are COL12A1 and COL14A1, and, of matrix only proteins, elastin (ELN). The proteomic signature thus revealed the similarities between but also some individual proteins of collagen membrane and matrix.

Abstract 13453: Estrogen Regulates Expression of Fibrotic Markers in Ips-derived Cardiac Fibroblasts and Cardiomyocytes

Introduction: Cardiovascular disease (CVD) is the leading cause of death among women. Epidemiologic studies indicate that pre-menopausal women are protected against the development of CVD when compared to age-matched men. Women show less cardiac remodeling, with downregulation in fibrosis and inflammation pathways. Remodeling is manifested by morphology and electrophysiological changes including hypertrophy, apoptosis, fibroblast proliferation, fibrosis and accumulation of fibrillar collagens. Hypothesis: We hypothesize that female protection against CVD is associated with estrogen levels as the incidence and severity of CVD increases after menopause. Methods: Induced pluripotent stem cells (IPSC) from male (M) and female (F) controls were differentiated to cardiomyocytes (IPSC-CMs) and cardiac fibroblasts (IPSC-CFs). Samples were cultured for 30 days and collected for analysis before and after 48 hs incubation with 1uM of 17-b-estradiol (EST). In IPSC-CMs we analyzed gene expression, morphology and beating frequency. Gene expression, morphology and wound healing were analyzed in IPSC-CFs. Results: Expression of ESR1 , and MYH7 was higher in female IPSC-CMs with upregulation after EST treatment in females but not males. Male IPSC-CMs showed higher expression of COL1A1 , CX43 , MYH6 and RYR2 ; MYH6 , RYR2 and COL1A1 were downregulated after EST treatment. No differences were observed in morphology or beating frequency before or after EST treatment (F:27.5±4.5 bpm vs M:22±2.5 bpm). In IPSC-CFs, males showed higher expression of the fibrotic markers COL1A1 , POSTN, SMAD2 and CCL2 ; EST treatment decreased the expression of POSTN and SMAD2 . Wound healing assays showed a decrease of the wound healing % in women after EST treatment (CT F:86.4±19.7% vs EST F:57.43±10.1%, at 8 hs, *p<0.05). Male wound healing rate was slower than women in control and treated groups, with no changes after EST treatment (CT F: 86.4±19.7% vs CT M: 51.65±18.4%, at 8 hs, *p<0.05). Conclusions: Estrogen treatment exerts an effect on gene expression in female and male IPSC-CMs and IPSC-CFs with men showing higher expression of fibrotic markers, which are down-regulated by EST. Further functional studies will shed light on the phenotypic effects of gene expression changes.

Distinct effects of different matrix proteoglycans on collagen fibrillogenesis and cell-mediated collagen reorganization

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.