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.

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.

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.

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.

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.

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.

A novel mouse model that develops spontaneous arthritis and is predisposed towards atherosclerosis.

OBJECTIVES: Patients with rheumatoid arthritis (RA) have a reduced life expectancy due to increased cardiovascular disease. The lack of a suitable animal model resembling both RA and atherosclerosis has hindered studies demonstrating a direct link between systemic inflammation in RA and the development of atherosclerosis. Our objective was to overcome this barrier by generating an animal model (K/BxA(g7)) that spontaneously develops both RA-like disease and atherosclerosis. METHODS: Arthritis severity was evaluated using clinical indices and immunohistochemical staining of ankle joint specimens. Aortic atherosclerosis was delineated via Sudan IV staining and immunohistochemical analysis. Serum cholesterol and lipoprotein levels were measured using enzymatic assays. Serum levels of cytokines, chemokines and adipokines were determined by Luminex assays. RESULTS: K/BxA(g7) mice developed a destructive arthropathy followed by prominent aortic atherosclerosis. These animals also displayed dyslipidaemia, characterised by reduced serum levels of total cholesterol and high-density lipoprotein, and increased low-density lipoprotein (LDL)/vLDL compared with control mice. Further, there were higher levels of circulating inflammatory mediators, such as interleukin-6, sRANKL and CCL5 in atherosclerotic K/BxA(g7) mice compared with controls. Treatment with etanercept reduced arthritis and atherosclerosis development in K/BxA(g7) mice. CONCLUSIONS: K/BxA(g7) mice recapitulate the same sequence of events occurring in patients with RA, namely an erosive, inflammatory arthritis followed by atherosclerosis. These data suggest that the K/BxA(g7) mouse is a novel system for investigating the interplay between systemic inflammation occurring in RA and the development of atherosclerosis.

Cardiomyocyte PAI-1 influences the cardiac transcriptome and limits the extent of cardiac fibrosis in response to left ventricular pressure overload.

Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid-acting inhibitor of endogenous plasminogen activators (uPA and tPA). The global PAI-1 knockout mice (PAI-1KO) develop age-dependent cardiac-selective fibrosis, and young global PAI-1KO mice exhibit augmented susceptibility to developing cardiac fibrosis in response to hypertension. Here, we tested the hypothesis that cardiomyocyte PAI-1 is necessary to provide cardioprotective effects in a left ventricular pressure overload-induced murine model of cardiac hypertrophy and fibrosis using cardiomyocyte-specific PAI-1 knockout (cmPAI-1KO) mice. The results revealed that cmPAI-1KO mice display significantly worse cardiac fibrosis than controls. To investigate the molecular mechanisms responsible for these effects, genome-wide cardiac transcriptome analysis was performed. Loss of cardiomyocyte PAI-1 led to differential expression of 978 genes compared to controls in response to left ventricular pressure overload. Pathway enrichment analysis identified the inflammatory response, cell substrate adhesion, regulation of cytokine production, leukocyte migration, extracellular matrix organization, and cytokine-mediated signaling pathways as being significantly upregulated in cmPAI-1KO hearts. Conversely, specific epigenetic repressors, cation transmembrane transport, muscle system processes, and nitric oxide signaling were significantly downregulated in cmPAI-1KO hearts compared to control hearts in response to left ventricular pressure overload. Collectively, the present study provides strong evidence of the impact of cardiomyocyte PAI-1 in regulation of the transcriptome network involved in the cardiac stress response. In response to stress, the deregulatory impact of cardiomyocyte PAI-1 loss on the cardiac transcriptome may be the underlying cause of cardiac-selective accelerated fibrogenesis in global PAI-1-deficient mice.

Pharmacological inhibition of plasminogen activator inhibitor-1 prevents memory deficits and reduces neuropathology in APP/PS1 mice.

RATIONALE: Extracellular proteolytic activity plays an important role in memory formation and the preservation of cognitive function. Previous studies have shown increased levels of plasminogen activator inhibitor-1 (PAI-1) in the brain of mouse models of Alzheimer's disease (AD) and plasma of AD patients, associated with memory and cognitive decline; however, the exact function of PAI-1 in AD onset and progression is largely unclear. OBJECTIVE: In this study, we evaluated a novel PAI-1 inhibitor, TM5A15, on its ability to prevent or reverse memory deficits and decrease Aß levels and plaque deposition in APP/PS1 mice. METHODS: We administered TM5A15 mixed in a chow diet to 3-month and 9-month-old APP/PS1 mice before and after neuropathological changes were distinguishable. We then evaluated the effects of TM5A15 on memory function and neuropathology at 9 months and 18 months of age. RESULTS: In the younger mice, 6 months of TM5A15 treatment protected against recognition and short-term working memory impairment. TM5A15 also decreased oligomer levels and amyloid plaques, and increased mBDNF expression in APP/PS1 mice at 9 months of age. In aged mice, 9 months of TM5A15 treatment did not significantly improve memory function nor decrease amyloid plaques. However, TM5A15 treatment showed a trend in decreasing oligomer levels in APP/PS1 mice at 18 months of age. CONCLUSION: Our results suggest that PAI-1 inhibition could improve memory function and reduce the accumulation of amyloid levels in APP/PS1 mice. Such effects are more prominent when TM5A15 is administered before advanced AD pathology and memory deficits occur.

A plasminogen activator inhibitor-1 inhibitor reduces airway remodeling in a murine model of chronic asthma.

We previously reported that plasminogen activator inhibitor (PAI)-1 deficiency prevents collagen deposition in the airways of ovalbumin (OVA)-challenged mice. In this study, we explored the therapeutic utility of blocking PAI-1 in preventing airway remodeling, using a specific PAI-1 inhibitor, tiplaxtinin. C57BL/6J mice were immunized with intraperitoneal injections of OVA on Days 0, 3, and 6. Starting on Day 11, mice were challenged with phosphate-buffered saline or OVA by nebulization three times per week for 4 weeks. Tiplaxtinin was mixed with chow and administered orally from 1 day before the phosphate-buffered saline or OVA challenge. Lung tissues were harvested after challenge and characterized histologically for infiltrating inflammatory cells, mucus-secreting goblet cells, and collagen deposition. Airway hyperresponsiveness was measured using whole-body plethysmography. Tiplaxtinin treatment significantly decreased levels of PAI-1 activity in bronchoalveolar lavage fluids, which indicates successful blockage of PAI-1 activity in the airways. The number of infiltrated inflammatory cells was reduced by tiplaxtinin treatment in the lungs of the OVA-challenged mice. Furthermore, oral administration of tiplaxtinin significantly attenuated the degree of goblet cell hyperplasia and collagen deposition in the airways of the OVA-challenged mice, and methacholine-induced airway hyperresponsiveness was effectively reduced by tiplaxtinin in these animals. This study supports our previous findings that PAI-1 promotes airway remodeling in a murine model of chronic asthma, and suggests that PAI-1 may be a novel target of treatment of airway remodeling in asthma.

Assessment of Virological Contributions to COVID-19 Outcomes in a Longitudinal Cohort of Hospitalized Adults.

BACKGROUND: While several demographic and clinical correlates of coronavirus disease 2019 (COVID-19) outcome have been identified, their relationship to virological and immunological parameters remains poorly defined. METHODS: To address this, we performed longitudinal collection of nasopharyngeal swabs and blood samples from a cohort of 58 hospitalized adults with COVID-19. Samples were assessed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load, viral genotype, viral diversity, and antibody titer. Demographic and clinical information, including patient blood tests and several composite measures of disease severity, was extracted from electronic health records. RESULTS: Several factors, including male sex, higher age, higher body mass index, higher 4C Mortality score, and elevated lactate dehydrogenase levels, were associated with intensive care unit admission. Of all measured parameters, only the retrospectively calculated median Deterioration Index score was significantly associated with death. While quantitative polymerase chain reaction cycle threshold (Ct) values and genotype of SARS-CoV-2 were not significantly associated with outcome, Ct value did correlate positively with C-reactive protein levels and negatively with D-dimer, lymphocyte count, and antibody titer. Intrahost viral genetic diversity remained constant through the disease course and resulted in changes in viral genotype in some participants. CONCLUSIONS: Ultimately, these results suggest that worse outcomes are driven by immune dysfunction rather than by viral load and that SARS-CoV-2 evolution in hospital settings is relatively constant over time.

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.

Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5.

Numerous studies have established that acute or chronic exposure to environmental pollutants like particulate matter (PM) leads to the development of accelerated aging related pathologies including pulmonary and cardiovascular diseases, and thus air pollution is one of the major global threats to human health. Air pollutant particulate matter 2.5 (PM2.5)-induced cellular dysfunction impairs tissue homeostasis and causes vascular and cardiopulmonary damage. To test a hypothesis that elevated plasminogen activator inhibitor-1 (PAI-1) levels play a pivotal role in air pollutant-induced cardiopulmonary pathologies, we examined the efficacy of a drug-like novel inhibitor of PAI-1, TM5614, in treating PM2.5-induced vascular and cardiopulmonary pathologies. Results from biochemical, histological, and immunohistochemical studies revealed that PM2.5 increases the circulating levels of PAI-1 and thrombin and that TM5614 treatment completely abrogates these effects in plasma. PM2.5 significantly augments the levels of pro-inflammatory cytokine interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF), and this also can be reversed by TM5614, indicating its efficacy in amelioration of PM2.5-induced increases in inflammatory and pro-thrombotic factors. TM5614 reduces PM2.5-induced increased levels of inflammatory markers cluster of differentiation 107 b (Mac3) and phospho-signal transducer and activator of transcription-3 (pSTAT3), adhesion molecule vascular cell adhesion molecule 1 (VCAM1), and apoptotic marker cleaved caspase 3. Longer exposure to PM2.5 induces pulmonary and cardiac thrombosis, but TM5614 significantly ameliorates PM2.5-induced vascular thrombosis. TM5614 also reduces PM2.5-induced increased blood pressure and heart weight. In vitro cell culture studies revealed that PM2.5 induces the levels of PAI-1, type I collagen, fibronectin (Millipore), and sterol regulatory element binding protein-1 and 2 (SREBP-1 and SREBP-2), transcription factors that mediate profibrogenic signaling, in cardiac fibroblasts. TM5614 abrogated that stimulation, indicating that it may block PM2.5-induced PAI-1 and profibrogenic signaling through suppression of SREBP-1 and 2. Furthermore, TM5614 blocked PM2.5-mediated suppression of nuclear factor erythroid related factor 2 (Nrf2), a major antioxidant regulator, in cardiac fibroblasts. Pharmacological inhibition of PAI-1 with TM5614 is a promising therapeutic approach to control air pollutant PM2.5-induced cardiopulmonary and vascular pathologies.

Role of PAI-1 in hepatic steatosis and dyslipidemia.

Plasminogen activator inhibitor 1 (PAI-1) is a functional biomarker of the metabolic syndrome. Previous studies have demonstrated that PAI-1 is a mechanistic contributor to several elements of the syndrome, including obesity, hypertension and insulin resistance. Here we show that PAI-1 is also a critical regulator of hepatic lipid metabolism. RNA sequencing revealed that PAI-1 directly regulates the transcriptional expression of numerous genes involved in mammalian lipid homeostasis, including PCSK9 and FGF21. Pharmacologic or genetic reductions in plasma PAI-1 activity ameliorates hyperlipidemia in vivo. These experimental findings are complemented with the observation that genetic deficiency of PAI-1 is associated with reduced plasma PCSK9 levels in humans. Taken together, our findings identify PAI-1 as a novel contributor to mammalian lipid metabolism and provides a fundamental mechanistic insight into the pathogenesis of one of the most pervasive medical problems worldwide.

PAI-1 contributes to homocysteine-induced cellular senescence.

Cellular Senescence is associated with organismal aging and related pathologies. Previously, we reported that plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of senescence and a potential therapeutic target for preventing aging-related pathologies. In this study, we investigate the efficacies of PAI-1 inhibitors in both in vitro and in vivo models of homocysteine (Hcy)-induced cardiovascular aging. Elevated Hcy, a known risk factor of cardiovascular diseases, induces endothelial senescence as evidenced by increased senescence-associated ß-Gal positivity (SA-ß-Gal), flattened cellular morphology, and cylindrical appearance of cellular nuclei. Importantly, inhibition of PAI-1 by small molecule inhibitors reduces the number of SA-ß-Gal positive cells, normalizes cellular morphology and nuclear shape. Furthermore, while Hcy induces the levels of senescence regulators PAI-1, p16, p53 and integrin ß3, and suppresses catalase expression, treatment with PAI-1 inhibitors blocks the Hcy-induced stimulation of senescence cadres, and reverses the Hcy-induced suppression of catalase, indicating that PAI-1 specific small molecule inhibitors are efficient to prevent Hcy-induced cellular senescence. Our in vivo study shows that the levels of integrin ß3, a recently identified potential regulator of cellular senescence, and its interaction with PAI-1 are significantly elevated in Hcy-treated heart tissues. In contrast, Hcy suppresses antioxidant gene regulator Nrf2 expression in hearts. However, co-treatment with PAI-1 inhibitor completely blocks the stimulation of Hcy-induced induction of integrin ß3 and reverses Nrf2 expression. Collectively these in vitro and in vivo studies indicate that pharmacological inhibition of PAI-1 improves endothelial and cardiac health by suppressing the pro-senescence effects of hyperhomocysteinemia through suppression of Hcy-induced master regulators of cellular senescence PAI-1 and integrin ß3. Therefore, PAI-1 inhibitors are promising drugs for amelioration of hyperhomocysteinemia-induced vascular aging and aging-related disease.