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Objective: Aberrant plasminogen activator inhibitor-1 (PAI-1) expression and activity have been implicated in bleeding disorders, multiorgan fibrosis, and wound healing anomalies. This study details the physiological consequences of targeted PAI-1 functional inhibition on cutaneous injury repair. Approach: Dorsal skin wounds from FVB/NJ mice, created with a 4 mm biopsy punch, were treated topically with the small-molecule PAI-1 antagonist tiplaxtinin (or vehicle control) for 5 days and then analyzed for markers of wound repair. Results: Compared to controls, tiplaxtinin-treated wounds displayed dramatic decreases in wound closure and re-epithelialization. PAI-1 immunoreactivity was evident at the migratory front in all injury sites indicating these effects were due to PAI-1 functional blockade and not PAI-1 expression changes. Stimulated HaCaT keratinocyte migration in response to recombinant PAI-1 in vitro was similarly attenuated by tiplaxtinin. While tiplaxtinin had no effect on keratinocyte proliferat...

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it’s cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a prominent member of the serine protease inhibitor superfamily (SERPIN) and a causative factor of multi-organ fibrosis as well as a key regulator of the tissue repair program. PAI-1 attenuates pericellular proteolysis by inhibiting the catalytic activity of both urokinase and tissue-type protease activators (uPA and tPA) effectively modulating, thereby, plasmin-mediated fibrinolysis and the overall pericellular proteolytic cascade. PAI-1 also impacts cellular responses to tissue injury and stress situations (growth, survival, migration) by titering the locale and temporal activation of multimeric cell-surface signaling complexes. This review will describe PAI-1 structure and function and detail the role of PAI-1 in the tissue repair program with an emphasis on cutaneous wound healing.

Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0 → G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5′-CACGTG-3′) at nucleotides −566 to −561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 “decoy” attenuated serum- and TGF-β1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67+ cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program. J. Cell. Biochem. 115: 1840–1847, 2014. © 2014 Wiley Periodicals, Inc.

Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels.

Multiple sclerosis (MS) is a chronic, debilitating condition mediated by inflammation and neurodegeneration. The ultimate goal of treatment is to delay or halt the progression of irreversible disability. Disease-modifying drugs (DMDs), including beta interferon and glatiramer acetate during phase III trails, have been shown to reduce relapse rates in relapsing-remitting multiple sclerosis (RRMS) as detected by magnetic resonance imaging (MRI). However, the long-term effects of DMDs on MS progression are not very clear; therefore, the aim of this paper is to evaluate the evidence available of the long-term effects of DMDs on reducing the progression of multiple sclerosis. A number of open-label, prospective extensions that followed a cohort of patients enrolled in double-blind, placebo-controlled trials were examined. Methodological difficulties faced in designing a trial of extended duration were hard to overcome, however, and long-term, open-label extensions of interferon and glatiramer acetate failed to show significant beneficial effects in delaying disability progression, questioning the cost-effectiveness of these therapies in the long-term.

Dhruba J Bharali, Marianne Khalil, Mujgan Gurbuz, Tessa M Simone, Shaker A MousaPharmaceutical Research Institute at Albany College of Pharmacy, Rensselaer, NY, USAAbstract: The emergence of nanotechnology has had a profound effect on many areas of healthcare and scientific research. Having grown exponentially, the focus of nanotechnology has been on engineering diversified novel applications that even go beyond therapeutic activity; nanotechnology also offers the ability to detect diseases, such as cancer, much earlier than ever imaginable. Often, patients diagnosed with breast, lung, colon, prostate, and ovarian cancer have hidden or overt metastatic colonies. With the advent of diagnostic nanotechnology, these numbers are expected to greatly diminish. This review provides a brief description of nanoparticle (liposome, quantum dot, and dendrimer)-mediated cancer therapy in the last decade with an emphasis on the development and use of dendrimers in cancer therapeutics.Keywords: nanoparticles, dendrimer, quantum dots, liposome

Metabolic syndrome places patients at high risk to many other health problems, as their bodies are in a perpetual state pro-inflammation and pro-thrombosis. In many cases, a regulated diet and increased exercise is sufficient in rectifying their condition, however for many patients, drug intervention is necessary. In recent years, calorie restriction has been shown to improve the health of patients afflicted with aging-related diseases, specifically those associated with metabolic syndrome, and researchers are now exploring drugs that mimic calorie restriction. Resveratrol mimics a calorie restricted diet as it has been shown to attenuate health benefits, including anti-platelet aggregation, vasorelaxation, atherosclerosis suppression, and lipid metabolism similar to a calorie restricted diet. Additionally, it modulates many of the pathways associated with metabolic syndrome, leading researchers to believe it has potential as a burgeoning therapeutic approach.

Direct-to-consumer advertising (DTCA) of drugs has been suggested to be a factor in the increased burden of healthcare spending within the US. This review article analyses the pharmaceutical spending differences between the US and New Zealand, two nations that allow DTCA. The pharmaceutical spending burden of New Zealand and the US was compared by assessing the impact of heavily advertised drugs and their position and rank in the pharmaceutical spending of their respective nation. The US spends far more money on pharmaceuticals than New Zealand. It may appear that heavily advertised drugs in the US have a potentially larger impact on what is being prescribed and paid for. It is also probable that the differences in healthcare systems in each nation (free market vs socialized medicine) can have an influence on pharmaceutical spending. The great amount of money being spent on pharmaceuticals per capita in the US is a more complex issue than can be solved solely by targeting DTCA.

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and the predominant physiologic inhibitor of urokinase (uPA) and tissue-type (tPA) plasmingen activators. This system effectively restricts, both spatially and temporally, the conversion of plasminogen to plasmin, thereby regulating physiologic and pathophysiologic stromal remodeling. Dysregulation of this cascade frequently results in anomalies of the tissue repair response. Elevated PAI-1 levels are a causative factor in various forms of vascular disease and tissue fibrotic syndromes. Independent of its role in proteolysis, PAI-1 stimulates cell motility via interacting with low-density lipoprotein receptor-related protein-1 (LRP1) activating several cellular signaling pathwaays. PAI-1 also regulates the availability of cell-surface integrins by promoting their endocytosis in an LRP-1- dependent manner via PAI-1/uPA/uPAR (uPA receptor)/LRPI/integrin complexes. This process fine tunes the special control of pericellular proteolysis and the overall cadence of cell detachment/re-adhesion required for efficient cell migration. These data suggest that PAI-1 modulates cell motility under several contexts, both via by its established anti-proteolytic properties and as a signaling initiator.

Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0 → G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5'-CACGTG-3') at nucleotides -566 to -561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 "decoy" attenuated serum- and TGF-β1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67(+) cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is the major physiologic regulator of the plasmin-based pericellular proteolytic cascade, a modulator of vascular smooth muscle cell (VSMC) migration and a causative factor in cardiovascular disease and restenosis, particularly in the context of increased vessel transforming growth factor- β1 (TGF-β1) levels. PAI-1 limits conversion of plasminogen to plasmin (and, thereby, fibrin degradation) by inhibiting its protease targets urokinase and tissue-type plasminogen activators (uPA, tPA). PAI-1 also has signaling functions and binds to the low density lipoprotein receptor-related protein 1 (LRP1) to regulate LRP1-dependent cell motility that, in turn, contributes to neointima formation. PAI-1/uPA/uPA receptor/LRPI/integrin complexes are endocytosed with subsequent uPAR/LRP1/integrin redistribution to the leading edge, initiating an "adhesion-detachment-readhesion" cycle to promote cell migration. PAI-1 also interacts with LRP1 in a uPA/uPAR-independent manner triggering Jak/Stat1 pathway activation to stimulate cell motility. PAI-1 itself is a substrate for extracellular proteases and exists in a "cleaved" form which, while unable to interact with uPA and tPA, retains LRP1-binding and migratory activity. These findings suggest that there are multiple mechanisms through which inhibition of PAI-1 may promote cardiovascular health. Several studies have focused on the design, synthesis and preclinical assessment of PAI-1 antagonists including monoclonal antibodies, peptides and low molecular weight (LMW) antagonists. This review discusses the translational impact of LMW PAI-1 antagonists on cardiovascular disease addressing PAI-1-initiated signaling, PAI-1 structure, the design and characteristics of PAI-1-targeting drugs, results of in vitro and in vivo studies, and their clinical implications.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a clade E1 member of the serine protease inhibitor (SERPIN) superfamily and the major physiologic inhibitor of the urokinase (uPA) and tissue-type (tPA) plasminogen activators. Elevated PAI-1 expression is a significant causative factor in vascular disease and a major contributor to the pathophysiology of diabetes, metabolic syndrome, stroke, atherosclerosis and restenosis, particularly in the setting of increased vessel TGF-β1 [1–3]. PAI-1 is unique relative to other SERPINs existing in the structurally and functionally distinct active, latent and cleaved conformations [4, 5]. PAI-1 is initially synthesized in an active state, capable of interacting with its proteinase targets, but is unstable (half-life of 2 hours at 37°C, pH 7.4) and converts spontaneously into a latent form [6]. Latency requires insertion of the N-terminus of the PAI-1 reactive center loop into β-sheet A forming a new β-strand (s4A) which creates an unusual loop structure and conformational change in the reactive center, ultimately preventing interaction with proteinases [7–9]. Alternatively, PAI-1 can be proteolytically-cleaved at the sissile P1-P1’ bond causing the N-terminal end of the reactive center loop to insert into β-sheet A, while the C-terminus of the reactive site loop forms strand s1C in β-sheet C. These structural rearrangments produce a 70A separation of the P1 and P1’ residues, thereby, preventing PAI-1 from complexing with the target proteinase due to spatial distortion, ultimately allowing for increased plasmin activation [10–12]. While neither cleaved nor latent PAI-1 forms complexes with their target proteases, all three conformations bind the low-density lipoprotein receptor-related protein-1 (LRP1) and initiate Jak/Stat signaling [13]. Elevated PAI-1 mRNA and protein expression are evident in the carotid vascular wall adjacent to thrombi induced by implantation of indwelling polyethylene tubing [14]. Furthermore, adenoviral delivery of PAI-1 potentiated neointima formation after catheter-induced injury while copper-stimulated neointima formation was reduced in PAI-1-null mice [15, 16]. In a mouse model of carotid artery ligation, PAI-1 protein levels are elevated in neointimal lesions 14-days after restriction. Regions expressing PAI-1 also express smooth muscle cell α-actin (Figure 1A,C), suggesting that PAI-1 is associated with smooth muscle cells (VSMCs). PAI-1 involvement in the pathological response to healing is reflected in its expression in the developing neointima in the ligated artery, but not the contralateral control vessel (Figure 1B,D), as well as in balloon-injured carotid arteries (Figure 1E). These findings implicate PAI-1 as a significant factor in the development of restenosis and provided the impetus for development of low-molecular weight PAI-1 antagonists. Tiplaxtinin (PAI-039), the most well studied small molecule PAI-1 inhibitor, attenuates asthmatic episodes, obesity, diabetes, cancer cell motility and angiogenesis [17–24]. The mechanism by which Tiplaxtinin antagonizes the anti-fibrinolytic activity of PAI-1 appears to involve inhibition of complex formation between PAI-1 and its target protease with promotion of PAI-1 cleavage [25, 26]. This has translational implications as PAI-1 deficiency in various cell types promotes plasmin-dependent apoptosis [27–31]. A decrease in PAI-1 antiproteolytic activity, through functional blockade or proteolytic cleavage, may subsequently increase VSMC apoptosis due to plasmin generation. One mechanism suggests that PAI-1 might contribute to neointimal growth by facilitating VSMC survival. Recent findings indicate that Tiplaxtinin induces VSMC apoptosis in a dose-dependent manner and this response was attenuated by the addition of TGF-β1. However, with the exception that PAI-1 binds and prevents the cleavage and activation of caspase-3, the role of PAI-1 in preventing VSMC apoptosis remains unexplored [31]. One attractive possibility is that PAI-1 might promote cell survival through the PI3K/Akt signaling axis and both PAI-1 and TGF- β1 stimulate AKT phosphorylation. Since PAI-1 is a highly upregulated gene in the TGF-β1 response set, TGF-β1 may activate Akt through PAI-1 or, at least, induce PAI-1 expression through an Akt-dependent pathway. Given the ubiquitous role PAI-1 plays in the etiology and progression of several chronic and acute fibrotic disorders, the therapeutic efficacy of small molecule PAI-1 inhibitors, such as Tiplaxtinin, may have translational adapatability beyond the scope of vascular disease. Figure 1 PAI-1 expression is upregulated within vascular smooth muscle cells of neointimal lesions

The organism's ability to regulate oxidative stress and metabolism is well recognized as a major determinant of longevity. While much research interest in this area is directed towards the study of genes that inhibit oxidative stress and/or improve metabolism, contribution to the aging process of genes with antagonistic effects on these two pathways is still less understood. The present study investigated the respective roles of the histone deacetylase Sirt1 and the thioredoxin binding protein TXNIP, two genes with opposite effects on oxidative stress and metabolism, in mediating the action of putative anti-aging interventions. Experiments were carried out in vitro and in vivo to determine the effect of proven, limited calorie availability, and unproven, resveratrol and dehydroepiandrosterone (DHEA), on the expression of Sirt1 and TXNIP. The results indicated that limited calorie availability consistently inhibited TXNIP in cancer and in normal cells including stem cells, however, it only slightly induced Sirt1expression in cancer cells. In contrast, resveratrol had a biphasic effect, and DHEA inhibited the expression of these two genes in a tissue specific manner, both in vitro and in vivo. Whereas all the three approaches tested inhibited TXNIP through the glycolytic pathway, DHEA acted by inhibiting G6PD and resveratrol through the activation of AMPK. In light of previous reports that Sirt1 induces AMPK-mediated signaling pathway, our findings point to the possibility of a negative relationship between Sirt1 and TXNIP that, if validated, can be exploited to improve the efficacy of putative anti-aging interventions.