MiR-125b Is Critical for Fibroblast-to-Myofibroblast Transition and Cardiac Fibrosis.

BACKGROUND: Cardiac fibrosis is the pathological consequence of stress-induced fibroblast proliferation and fibroblast-to-myofibroblast transition. MicroRNAs have been shown to play a central role in the pathogenesis of cardiac fibrosis. We identified a novel miRNA-driven mechanism that promotes cardiac fibrosis via regulation of multiple fibrogenic pathways. METHODS AND RESULTS: Using a combination of in vitro and in vivo studies, we identified that miR-125b is a novel regulator of cardiac fibrosis, proliferation, and activation of cardiac fibroblasts. We demonstrate that miR-125b is induced in both fibrotic human heart and murine models of cardiac fibrosis. In addition, our results indicate that miR-125b is necessary and sufficient for the induction of fibroblast-to-myofibroblast transition by functionally targeting apelin, a critical repressor of fibrogenesis. Furthermore, we observed that miR-125b inhibits p53 to induce fibroblast proliferation. Most importantly, in vivo silencing of miR-125b by systemic delivery of locked nucleic acid rescued angiotensin II-induced perivascular and interstitial fibrosis. Finally, the RNA-sequencing analysis established that miR-125b altered the gene expression profiles of the key fibrosis-related genes and is a core component of fibrogenesis in the heart. CONCLUSIONS: In conclusion, miR-125b is critical for induction of cardiac fibrosis and acts as a potent repressor of multiple anti-fibrotic mechanisms. Inhibition of miR-125b may represent a novel therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Nitric oxide prevents alveolar senescence and emphysema in a mouse model.

Nω-nitro-L-arginine methyl ester (L-NAME) treatment induces arteriosclerosis and vascular senescence. Here, we report that the systemic inhibition of nitric oxide (NO) production by L-NAME causes pulmonary emphysema. L-NAME-treated lungs exhibited both the structural (alveolar tissue destruction) and functional (increased compliance and reduced elastance) characteristics of emphysema development. Furthermore, we found that L-NAME-induced emphysema could be attenuated through both genetic deficiency and pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is an important contributor to the development of senescence both in vitro and in vivo, we investigated whether L-NAME-induced senescence led to the observed emphysematous changes. We found that L-NAME treatment was associated with molecular and cellular evidence of premature senescence in mice, and that PAI-1 inhibition attenuated these increases. These findings indicate that NO serves to protect and defend lung tissue from physiological aging.

PAI-1-regulated extracellular proteolysis governs senescence and survival in Klotho mice.

Cellular senescence restricts the proliferative capacity of cells and is accompanied by the production of several proteins, collectively termed the "senescence-messaging secretome" (SMS). As senescent cells accumulate in tissue, local effects of the SMS have been hypothesized to disrupt tissue regenerative capacity. Klotho functions as an aging-suppressor gene, and Klotho-deficient (kl/kl) mice exhibit an accelerated aging-like phenotype that includes a truncated lifespan, arteriosclerosis, and emphysema. Because plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor (SERPIN), is elevated in kl/kl mice and is a critical determinant of replicative senescence in vitro, we hypothesized that a reduction in extracellular proteolytic activity contributes to the accelerated aging-like phenotype of kl/kl mice. Here we show that PAI-1 deficiency retards the development of senescence and protects organ structure and function while prolonging the lifespan of kl/kl mice. These findings indicate that a SERPIN-regulated cell-nonautonomous proteolytic cascade is a critical determinant of senescence in vivo.

Plasminogen activator inhibitor-1 antagonist TM5441 attenuates Nω-nitro-L-arginine methyl ester-induced hypertension and vascular senescence.

BACKGROUND: Long-term inhibition of nitric oxide synthase by L-arginine analogues such as N(ω)-nitro-l-arginine methyl ester (L-NAME) has been shown to induce senescence in vitro and systemic hypertension and arteriosclerosis in vivo. We previously reported that plasminogen activator inhibitor-1 (PAI-1)-deficient mice (PAI-1(-/-)) are protected against L-NAME-induced pathologies. In this study, we investigated whether a novel, orally active PAI-1 antagonist (TM5441) has a similar protective effect against L-NAME treatment. Additionally, we studied whether L-NAME can induce vascular senescence in vivo and investigated the role of PAI-1 in this process. METHODS AND RESULTS: Wild-type mice received either L-NAME or L-NAME and TM5441 for 8 weeks. Systolic blood pressure was measured every 2 weeks. We found that TM5441 attenuated the development of hypertension and cardiac hypertrophy compared with animals that had received L-NAME alone. Additionally, TM5441-treated mice had a 34% reduction in periaortic fibrosis relative to animals on L-NAME alone. Finally, we investigated the development of vascular senescence by measuring p16(Ink4a) expression and telomere length in aortic tissue. We found that L-NAME increased p16(Ink4a) expression levels and decreased telomere length, both of which were prevented with TM5441 cotreatment. CONCLUSIONS: Pharmacological inhibition of PAI-1 is protective against the development of hypertension, cardiac hypertrophy, and periaortic fibrosis in mice treated with L-NAME. Furthermore, PAI-1 inhibition attenuates the arterial expression of p16(Ink4a) and maintains telomere length. PAI-1 appears to play a pivotal role in vascular senescence, and these findings suggest that PAI-1 antagonists may provide a novel approach in preventing vascular aging and hypertension.

Global gene expression profiling in PAI-1 knockout murine heart and kidney: molecular basis of cardiac-selective fibrosis.

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1 (PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed.

Enhanced functional stability of plasminogen activator inhibitor-1 in patients with livedoid vasculopathy.

Livedoid vasculopathy (LV) is a chronic, recurrent, painful cutaneous disease with distinctive clinical features and an uncertain etiology. The skin lesions are recognizable by focal purpura, depigmentation and shallow ulcers. Thrombophilic conditions occur frequently in patients with LV. While no definitive treatment exists for LV, smoking cessation, antiplatelet therapy, immunosuppressive treatment, and anabolic steroids are often included in the therapeutic ladder. Recently, a possible link between LV and impaired fibrinolysis was established as cutaneous LV lesions responded to tissue plasminogen activator (t-PA) infusion suggesting that inhibition of the fibrinolysis through plasminogen activator inhibitor-1 (PAI-1) activity may determine the disease course in patients with LV. In this study, we investigated PAI-1 antigen (Ag) and activity levels in 20 patients with biopsy proven LV (mean age 26 ± 11, M/F = 7/13, median disease duration 3.5 years). All patients received antiplatelet treatment with aspirin and/or dipyrimadole and 14 patients received anabolic steroids or immunosuppressive treatment. Fasting PAI-1 Ag and activity levels were measured at 9 AM in all patients. Both Ag (34 (26) ng/ml) (median (interquartile range)) and specific activity (17 (23) IU/fmole) levels of PAI-1 were moderately elevated in LV patients compared to the controls, however, PAI-1 kinetic studies demonstrated markedly enhanced stability of PAI-1 activity in plasma from patients with LV. Specific activity at 16 h was significantly higher than expected specific activity levels (7 (11) vs. 0.07 (0.09) IU/fmole, P < 0.01). While the exact mechanism of increased stability of PAI-1 activity is not known, it may be due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of LV, and systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative in patients with LV.

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.