Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells.

Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI-1 activity, for CML patients.

Structural Insight into the Two-Step Mechanism of PAI-1 Inhibition by Small Molecule TM5484.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is the main physiological inhibitor of plasminogen activators. By interacting with matrix components, including vitronectin (Vn), PAI-1 plays a regulatory role in tissue remodeling, cell migration, and intracellular signaling. Emerging evidence points to a role for PAI-1 in various pathological conditions, including cardiovascular diseases, cancer, and fibrosis. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A class of small molecule inhibitors including TM5441 and TM5484, designed to bind the cleft in the central ß-sheet A of PAI-1, showed to be potent PAI-1 inhibitors in vivo. However, their binding site has not yet been confirmed. Here, we report two X-ray crystallographic structures of PAI-1 in complex with TM5484. The structures revealed a binding site at the flexible joint region, which is distinct from the presumed binding site. Based on the structural analysis and biochemical data we propose a mechanism for the observed dose-dependent two-step mechanism of PAI-1 inhibition. By binding to the flexible joint region in PAI-1, TM5484 might restrict the structural flexibility of this region, thereby inducing a substrate form of PAI-1 followed by a conversion to an inert form.

Effects of lisinopril treatment on the pathophysiology of PCOS and plasminogen activator inhibitor-1 concentrations in rats.

RESEARCH QUESTION: Angiotensin-converting enzyme inhibition results in a significant reduction in plasma concentrations of plasminogen activator inhibitor-1 (PAI-1). What are the effects of lisinopril treatment on PAI-1 concentrations and the morphology and function of the ovaries in the letrozole-induced polycystic ovary syndrome (PCOS) rat model? DESIGN: This prospective randomized controlled animal study involved female Wistar albino rats. Twelve rats were assigned as controls (group I). In the study group (n = 48), letrozole (an aromatase inhibitor) was administered for PCOS modelling for 9 weeks. After confirming disrupted oestrous cycles, the study group was randomized into two groups: group II (n = 24; letrozole only) and group III (n = 24; letrozole + lisinopril 15 mg/kg per day). After 12 weeks, each group was divided randomly into two. Biochemical, histopathological and immunohistochemical analyses was performed in subgroups designated A, and fertilization rates were studied in subgroups designated B. RESULTS: Lisinopril treatment reduced the weight and area of the ovaries, the number and wall thickness of cystic follicles, and serum concentrations of LH and testosterone, relative to group II (P < 0.001). Circulating PAI-1 concentrations were significantly different among three groups (7.7 ± 0.9 ng/ml, 9.8 ± 0.7 ng/ml and 8.6 ± 0.7 ng/ml for groups IA, IIA and IIIA; P < 0.001). Pregnancy rates were 100%, 0% and 16.7% in groups IB, IIB and IIIB. CONCLUSIONS: In the letrozole-induced rodent PCOS model, lisinopril modifies the action of letrozole, possibly by inhibition of systemic and ovarian production of PAI-1. The use of PAI-1 inhibitors deserves further investigation in understanding the pathogenesis of PCOS.

Association between plasminogen activator inhibitor-1 in young adulthood and nonalcoholic fatty liver disease in midlife: CARDIA.

BACKGROUND: Prior studies have demonstrated a cross-sectional association between elevated plasminogen activator inhibitor-1 (PAI-1) levels and nonalcoholic fatty liver disease (NAFLD). However, there are no prospective longitudinal assessments of the association between PAI-1 and NAFLD. We aimed to describe the association between PAI-1 levels in early adulthood with NAFLD in midlife. METHODS: Among the 5115 participants in the coronary artery risk development in young adults (CARDIA) study, participants were randomly selected from a subset that was free of obesity, diabetes and hypertension at the 1992-1993 exam and attended the 2005-2006 exam (n = 996). A subset of participants (n = 896) also had CT liver fat measured (2010-2011). Participants with secondary causes of steatosis were excluded (n = 87). NAFLD was defined as liver attenuation ≤51 Hounsfield units. Logistic regression models assessed the association between PAI-1 and NAFLD. RESULTS: Of 809 participants, 53% were female, 37% black with a mean age of 32 years. Median PAI-1 level at 1st assessment (1992-1993) was 23.4 ng/mL among participants with NAFLD vs 11.9 ng/mL among those without NAFLD (P < .0001). Median PAI-1 level at 2nd assessment (2005-2006) was 55.6 ng/mL among participants with NAFLD vs 19.5 ng/mL among those without NAFLD (P < .0001). Higher PAI-1 levels were independently associated with NAFLD (1st assessment adjusted OR [AOR] 2.16 per 1 standard deviation higher log(PAI-1) level (95% confidence interval [CI] 1.63-2.85); 2nd assessment AOR 2.71 (95% CI 2.03-3.61)). CONCLUSIONS: Plasma PAI-1 levels in young adulthood were independently associated with NAFLD in midlife. Further studies may indicate whether PAI-1 plays a role in NAFLD pathophysiology.

Acetyltransferase p300 inhibitor reverses hypertension-induced cardiac fibrosis.

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension-induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300-specific small molecule inhibitor L002 or C646 reverses hypertension-induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension-induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension-induced cardiac hypertrophy and fibrosis, and discover new anti-hypertrophic and anti-fibrotic candidates.

TGF-ß-induced intracellular PAI-1 is responsible for retaining hematopoietic stem cells in the niche.

Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-ß-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-ß signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-ß-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-ß-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.

Fundamental Cardiovascular Research: Returns on Societal Investment: A Scientific Statement From the American Heart Association.

Recent decades have witnessed robust successes in conquering the acutely lethal manifestations of heart and vascular diseases. Many patients who previously would have died now survive. Lifesaving successes like these provide a tremendous and easily recognized benefit to individuals and society. Although cardiovascular mortality has declined, the devastating impact of chronic heart disease and comorbidities on quality of life and healthcare resources continues unabated. Future strides, extending those made in recent decades, will require continued research into mechanisms underlying disease prevention, pathogenesis, progression, and therapeutic intervention. However, severe financial constraints currently jeopardize these efforts. To chart a path for the future, this report analyzes the challenges and opportunities we face in continuing the battle against cardiovascular disease and highlights the return on societal investment afforded by fundamental cardiovascular research.

Plasminogen Activator Inhibitor Type I Controls Cardiomyocyte Transforming Growth Factor-ß and Cardiac Fibrosis.

BACKGROUND: Fibrosis is the pathological consequence of stress-induced tissue remodeling and matrix accumulation. Increased levels of plasminogen activator inhibitor type I (PAI-1) have been shown to promote fibrosis in multiple organ systems. Paradoxically, homozygous genetic deficiency of PAI-1 is associated with spontaneous age-dependent, cardiac-selective fibrosis in mice. We have identified a novel PAI-1-dependent mechanism that regulates cardiomyocyte-derived fibrogenic signals and cardiac transcriptional pathways during injury. METHODS: Cardiac fibrosis in subjects with homozygous mutation in SERPINE-1 was evaluated with late gadolinium-enhanced cardiac magnetic resonance imaging. A murine cardiac injury model was performed by subcutaneous infusion of either saline or Angiotensin II by osmotic minipumps. We evaluated blood pressure, cardiac function (by echocardiography), fibrosis (with Masson Trichrome staining), and apoptosis (with TUNEL staining), and we performed transcriptome analysis (with RNA sequencing). We further evaluated fibrotic signaling in isolated murine primary ventricular myocytes. RESULTS: Cardiac fibrosis was detected in 2 otherwise healthy humans with complete PAI-1 deficiency because of a homozygous frameshift mutation in SERPINE-1. In addition to its suppressive role during spontaneous cardiac fibrosis in multiple species, we hypothesized that PAI-1 also regulates fibrosis during cardiac injury. Treatment of young PAI-1-/- mice with Angiotensin II induced extensive hypertrophy and fibrotic cardiomyopathy, with increased cardiac apoptosis and both reactive and replacement fibrosis. Although Angiotensin II-induced hypertension was blunted in PAI-1-/- mice, cardiac hypertrophy was accelerated. Furthermore, ventricular myocytes were found to be an important source of cardiac transforming growth factor-ß (TGF-ß) and PAI-1 regulated TGF-ß synthesis by cardiomyocytes in vitro as well as in vivo during cardiac injury. Transcriptome analysis of ventricular RNA after Angiotensin II treatment confirmed that PAI-1 deficiency significantly enhanced multiple TGF-ß signaling elements and transcriptional targets, including genes for extracellular matrix components, mediators of extracellular matrix remodeling, matricellular proteins, and cardiac integrins compared with wild-type mice. CONCLUSIONS: PAI-1 is an essential repressor of cardiac fibrosis in mammals. We define a novel cardiomyocyte-specific regulatory mechanism for TGF-ß production by PAI-1, which explains the paradoxical effect of PAI-1 deficiency in promoting cardiac-selective fibrosis. Thus, PAI-1 is a molecular switch that controls the cardiac TGF-ß axis and its early transcriptional effects that lead to myocardial fibrosis.

Plasminogen Activator Inhibitor-1 Is a Marker and a Mediator of Senescence.

PAI-1 (plasminogen activator inhibitor-1) is a member of the evolutionarily conserved serine protease inhibitor family and a potent and rapid-acting inhibitor of both of the mammalian plasminogen activators. Organismal homeostasis requires physiological levels of endogenous PAI-1, and increased PAI-1 production guides the onset and progression of numerous human diseases and contributes to the multimorbidity of aging. Both chronological and stress-induced accelerated aging are associated with cellular senescence and accompanied by marked increases in PAI-1 expression in tissues. Recent studies suggest that PAI-1 is not only a marker but also a key mediator of cellular senescence and organismal aging. Here, we review the significance of PAI-1 as a bonafide marker, as well as a critical mediator, of cellular senescence associated with aging and aging-related pathologies.

Epigenetics in Reactive and Reparative Cardiac Fibrogenesis: The Promise of Epigenetic Therapy.

Epigenetic changes play a pivotal role in the development of a wide spectrum of human diseases including cardiovascular diseases, cancer, diabetes, and intellectual disabilities. Cardiac fibrogenesis is a common pathophysiological process seen during chronic and stress-induced accelerated cardiac aging. While adequate production of extracellular matrix (ECM) proteins is necessary for post-injury wound healing, excessive synthesis and accumulation of extracellular matrix protein in the stressed or injured hearts causes decreased or loss of lusitropy that leads to cardiac failure. This self-perpetuating deposition of collagen and other matrix proteins eventually alter cellular homeostasis; impair tissue elasticity and leads to multi-organ failure, as seen during pathogenesis of cardiovascular diseases, chronic kidney diseases, cirrhosis, idiopathic pulmonary fibrosis, and scleroderma. In the last 25 years, multiple studies have investigated the molecular basis of organ fibrosis and highlighted its multi-factorial genetic, epigenetic, and environmental regulation. In this minireview, we focus on five major epigenetic regulators and discuss their central role in cardiac fibrogenesis. Additionally, we compare and contrast the epigenetic regulation of hypertension-induced reactive fibrogenesis and myocardial infarction-induced reparative or replacement cardiac fibrogenesis. As microRNAs-one of the major epigenetic regulators-circulate in plasma, we also advocate their potential diagnostic role in cardiac fibrosis. Lastly, we discuss the evolution of novel epigenetic-regulating drugs and predict their clinical role in the suppression of pathological cardiac remodeling, cardiac aging, and heart failure. J. Cell. Physiol. 232: 1941-1956, 2017. © 2016 Wiley Periodicals, Inc.

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.

Klotho, an antiaging molecule, attenuates oxidant-induced alveolar epithelial cell mtDNA damage and apoptosis.

Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing (MCAT) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

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.

ApoE deficiency exacerbates the development and sustainment of a semi-chronic K/BxN serum transfer-induced arthritis model.

BACKGROUND: The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE. METHODS: K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE(-/-) mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively. RESULTS: ApoE(-/-) mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE(-/-) mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE(-/-) mice. However, arthritic ApoE(-/-) mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE(-/-) mice and arthritic C57BL/6 mice. CONCLUSIONS: Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis.

DNA Damage: A Main Determinant of Vascular Aging.

Vascular aging plays a central role in health problems and mortality in older people. Apart from the impact of several classical cardiovascular risk factors on the vasculature, chronological aging remains the single most important determinant of cardiovascular problems. The causative mechanisms by which chronological aging mediates its impact, independently from classical risk factors, remain to be elucidated. In recent years evidence has accumulated that unrepaired DNA damage may play an important role. Observations in animal models and in humans indicate that under conditions during which DNA damage accumulates in an accelerated rate, functional decline of the vasculature takes place in a similar but more rapid or more exaggerated way than occurs in the absence of such conditions. Also epidemiological studies suggest a relationship between DNA maintenance and age-related cardiovascular disease. Accordingly, mouse models of defective DNA repair are means to study the mechanisms involved in biological aging of the vasculature. We here review the evidence of the role of DNA damage in vascular aging, and present mechanisms by which genomic instability interferes with regulation of the vascular tone. In addition, we present potential remedies against vascular aging induced by genomic instability. Central to this review is the role of diverse types of DNA damage (telomeric, non-telomeric and mitochondrial), of cellular changes (apoptosis, senescence, autophagy), mediators of senescence and cell growth (plasminogen activator inhibitor-1 (PAI-1), cyclin-dependent kinase inhibitors, senescence-associated secretory phenotype (SASP)/senescence-messaging secretome (SMS), insulin and insulin-like growth factor 1 (IGF-1) signaling), the adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-nuclear factor kappa B (NFκB) axis, reactive oxygen species (ROS) vs. endothelial nitric oxide synthase (eNOS)-cyclic guanosine monophosphate (cGMP) signaling, phosphodiesterase (PDE) 1 and 5, transcription factor NF-E2-related factor-2 (Nrf2), and diet restriction.

Enhanced efferocytosis of apoptotic cardiomyocytes through myeloid-epithelial-reproductive tyrosine kinase links acute inflammation resolution to cardiac repair after infarction.

RATIONALE: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. OBJECTIVE: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. METHODS AND RESULTS: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6c(LO) myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk(+/+) marrow into Mertk(-/-) mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. CONCLUSIONS: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

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.

Role of plasminogen activator inhibitor-1 in senescence and aging.

The average age of the US population continues to increase. Age is the most important determinant of disease and disability in humans, but the fundamental mechanisms of aging remain largely unknown. Many age-related diseases are associated with an impaired fibrinolytic system. Elevated plasminogen activator inhibitor-1 (PAI-1) levels are reported in age-associated clinical conditions including cardiovascular diseases, type 2 diabetes, obesity and inflammation. PAI-1 levels are also elevated in animal models of aging. While the association of PAI-1 with physiological aging is well documented, it is only recently that its critical role in the regulation of aging and senescence has become evident. PAI-1 is synthesized and secreted in senescent cells and contributes directly to the development of senescence by acting downstream of p53 and upstream of insulin-like growth factor binding protein-3. Pharmacologic inhibition or genetic deficiency of PAI-1 was shown to be protective against senescence and the aging-like phenotypes in kl/kl and N(ω)-nitro-l-arginine methyl ester-treated wild-type mice. Further investigation into PAI-1's role in senescence and aging will likely contribute to the prevention and treatment of aging-related pathologies.

A small molecule inhibitor to plasminogen activator inhibitor 1 inhibits macrophage migration.

OBJECTIVE: Macrophage (Mϕ) migration rests on the adhesion/detachment between Mϕ surface components and extracellular matrixes, and the contribution of numerous inflammatory disorders. Plasminogen activator inhibitor (PAI)-1, a serine protease inhibitor, influences Mϕ motility through an action distinct from its classical modulation of the plasmin-based fibrinolytic process. We rely here on a small molecule PAI-1 inhibitor (TM5275) to investigate the role of PAI-1 in Mϕ migration in the pathogenesis of renal injury. APPROACH AND RESULTS: Mϕ migration was inhibited both in vitro and in vivo by TM5275. It was also reduced in T-cell-deficient nude mice, but not in PAI-1-deficient mice. Mϕ migration hinged on the interaction of PAI-1 with low-density lipoprotein receptor-related protein, an interaction prevented by TM5275, but not with vitronectin, urokinase-type plasminogen activator, or tissue-type plasminogen activator. Fed to rats with anti-Thy-1-induced nephritis, TM5275 significantly decreased Mϕ accumulation and ameliorated the progression of renal injury. CONCLUSIONS: These findings suggest that a small molecule PAI-1 inhibitor represents a novel class of anti-inflammatory agents targeting Mϕ migration by the inhibition of the interaction of PAI-1 with low-density lipoprotein receptor-related protein.

Functionally stable plasminogen activator inhibitor-1 in a family with cardiovascular disease and vitiligo.

Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely 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 vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development.