Molecular basis of cardiac endothelial-to-mesenchymal transition (EndMT): differential expression of microRNAs during EndMT.

Fibroblasts are responsible for producing the majority of collagen and other extracellular matrix (ECM) proteins in tissues. In the injured tissue, transforming growth factor-ß (TGF-ß)-activated fibroblasts or differentiated myofibroblasts synthesize excessive ECM proteins and play a pivotal role in the pathogenesis of fibrosis in heart, kidney and other organs. Recent studies suggest that fibroblast-like cells, derived from endothelial cells by endothelial-to-mesenchymal transition (EndMT), contribute to the pathogenesis of cardiac fibrosis. The molecular basis of EndMT, however, is poorly understood. Here, we investigated the molecular basis of EndMT in mouse cardiac endothelial cells (MCECs) in response to TGF-ß2. MCECs exposed to TGF-ß2 underwent EndMT as evidenced by morphologic changes, lack of acetylated-low density lipoprotein (Ac-LDL) uptake, and the presence of alpha-smooth muscle actin (α-SMA) staining. Treatment with SB431542, a small molecule inhibitor of TGF-ß-receptor I (TßRI) kinase, but not PD98059, a MEK inhibitor, completely blocked TGF-ß2-induced EndMT. The transcript and protein levels of α-SMA, Snail and ß-catenin as well as acetyltransferase p300 (ATp300) were elevated in EndMT derived fibroblast-like cells. Importantly, microRNA (miRNA) array data revealed that the expression levels of specific miRNAs, known to be dysregulated in different cardiovascular diseases, were altered during EndMT. The protein level of cellular p53, a bonafide target of miR-125b, was downregulated in EndMT-derived fibroblast-like cells. Here, we report for the first time, the differential expression of miRNAs during cardiac EndMT. These results collectively suggest that TßRI serine-threonine kinase-induced TGF-ß signaling and microRNAs, the epigenetic regulator of gene expression at the posttranscriptional level, are involved in EndMT and promote profibrotic signaling in EndMT-derived fibroblast-like cells. Pharmacologic agents that restrict the progression of cardiac EndMT, a phenomenon that is found in adults only in the pathological conditions, in targeting specific miRNA may be helpful in preventing and treating cardiac fibrosis.

Genetic deficiency of plasminogen activator inhibitor-1 promotes cardiac fibrosis in aged mice: involvement of constitutive transforming growth factor-beta signaling and endothelial-to-mesenchymal transition.

BACKGROUND: Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1-deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis. METHODS AND RESULTS: Histological evaluation of myocardial tissues derived from aged PAI-1-deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-ß1/2 and the number of Mac3-positive and fibroblast specific protein-1-positive cells were significantly elevated in aged PAI-1-deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1-deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1-deficient mice. The numbers of phosphorylated Smad2-, phosphorylated Smad3-, and phosphorylated ERK1/2 MAPK-, but not pAkt/PKB-, positive cells were significantly increased in PAI-1-deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1-deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-ß2. CONCLUSIONS: These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-ß signaling may contribute to profibrotic responses in aged PAI-1-deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1-deficient mice.

Expression and regulation of soluble epoxide hydrolase in adipose tissue.

Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a "western diet." sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3-L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a "western diet," total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increased the expression of sEH in mature 3T3-L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.

Macrophage TNF-alpha contributes to insulin resistance and hepatic steatosis in diet-induced obesity.

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-alpha. To test this hypothesis, we transplanted male wild-type (WT) and TNF-alpha deficient (KO) mice with either TNF-alpha-sufficient (TNF-alpha(+/+)) or TNF-alpha-deficient (TNF-alpha(-/-)) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-alpha, which was partially explained by reduced epididymal fat pad and liver weight. TNF-alpha(-/-) -->KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-alpha(+/+)-->KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-alpha(-/-) bone marrow in WT mice compared with TNF-alpha(+/+)-->WT. The preserved insulin sensitivity seen in TNF-alpha(-/-)-->KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-alpha contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-alpha.

Bone marrow plasminogen activator inhibitor-1 influences the development of obesity.

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.

Differential and opposing regulation of PAI-1 promoter activity by estrogen receptor alpha and estrogen receptor beta in endothelial cells.

To investigate the molecular mechanisms involved in the estrogen-dependent control of plasminogen activator inhibitor-1 (PAI-1) gene expression in vascular cells, we compared the transactivation properties of estrogen receptors (ERalpha and ERbeta) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ERalpha increased PAI-1 promoter activity in BAECs by an estrogen-dependent mechanism, whereas ERbeta suppressed PAI-1 promoter activity by an estrogen-independent mechanism. The suppressive activity of ERbeta was dominant over the inductive activity of ERalpha. Mutation of a putative estrogen response element (ERE) located at position -427 in the proximal promoter abolished the ERalpha action without influencing the suppressive effects of ERbeta. Mutation of either AP1-like site did not eliminate the ERalpha or ERbeta actions at the PAI-1 promoter, suggesting that other promoter elements are involved in these responses. These mutations significantly reduced the -3.4kbp PAI-1 promoter response to serum. We concluded that ERalpha and ERbeta exert differential effects on the PAI-1 promoter activity in transfected BAECs. ERalpha activated the PAI-1 promoter through a proximal ERE (-427) and possibly additional EREs located within the PAI-1 promoter, whereas ERbeta suppressed the promoter construct via an unidentified mechanism. This is the first demonstration of the differential regulation of a vascular gene promoter by ERalpha and ERbeta.

Tumor necrosis factor alpha activates the human plasminogen activator inhibitor-1 gene through a distal nuclear factor kappaB site.

Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of plasminogen activation and likely plays important roles in coronary thrombosis and arteriosclerosis. Tumor necrosis factor-alpha (TNFalpha) is one of many recognized physiological regulators of PAI-1 expression and may contribute to elevated plasma PAI-1 levels in sepsis and obesity. Although TNFalpha is a potent inducer of PAI-1 expression in vitro and in vivo, the precise location of the TNFalpha response site in the PAI-1 promoter has yet to be determined. Transient transfection studies using luciferase reporter constructs containing PAI-1 promoter sequence up to 6.4 kb failed to detect a response to TNFalpha. Moreover, TNFalpha failed to induce expression of enhanced green fluorescent protein under the control of a 2.9-kb human PAI-1 promoter in transgenic mice, although endogenous murine PAI-1 was strongly induced. These data suggested that the TNFalpha response element in the PAI-1 gene is remote from the proximal promoter region. In this study, seven candidate regulatory regions were identified using cross-species sequence homology analysis as well as DNase I-hypersensitive site analysis. We identified a 5' distal TNFalpha-responsive enhancer of the PAI-1 gene located 15 kb upstream of the transcription start site containing a conserved NFkappaB-binding site that mediates the response to TNFalpha. This newly recognized site is fully capable of binding NFkappaB subunits p50 and p65, whereas overexpression of the NFkappaB inhibitor IkappaB prevents TNFalpha-induced activation of this enhancer element.

Ligand-specific control of src-suppressed C kinase substrate gene expression.

The src-suppressed C-kinase substrate, SSeCKS, is now recognized as a key regulator of cell signaling and cytoskeletal dynamics. However, few ligands that control SSeCKS expression have been identified. We report that platelet-derived growth factor-BB (PDGF-BB), lysophosphatidic acid (LPA), and eicosapentaenoic acid (EPA) potently modulate SSeCKS gene expression in cultured smooth muscle (RASM) cells relative to other bioactive ligands tested. In addition, EPA-dependent regulation of SSeCKS expression correlates with distinct changes in cell morphology and adhesion in RASM cells. Independent evidence that ligand-specific control of SSeCKS expression links to the regulation of cell adhesion and morphology was obtained using ras-transformed fibroblasts, KNRK. Sodium butyrate (NaB) upregulates SSeCKS mRNA and protein expression corresponding to increased cell-spreading and adhesion. In addition, ectopic expression of recombinant SSeCKS recapitulates attributes of NaB-induced morphogenesis in KNRK cells. The data provide novel evidence that SSeCKS functions in PDGF-BB-, LPA-, EPA-, and NaB-mediated cell signaling.