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Degradation and resynthesis of the extracellular matrix (ECM) are essential during tissue remodeling. Expansion of the vascular intima in atherosclerosis and restenosis following injury is dependent upon smooth muscle cell (SMC) proliferation and migration. The migration of SMC from media to intima critically depends on degradation of ECM protein by matrix metalloproteinases (MMPs). MMP inhibitors and eNOS gene transfer have been shown to inhibit SMC migration in vitro and neointima formation in vivo. Nitric oxide (NO) and cyclic-GMP have been implicated in the inhibition of VSMC migration. But, there are few studies addressing the role of NO signaling pathways on the expression of MMPs. Here we reported the involvement of cyclic-GMP-dependent protein kinase (PKG) (an important mediator of NO and cGMP signaling pathway in VSMC) on MMP-2 expression in rat aortic SMC. The goal of the present study was to gain insight into the possible involvement of PKG on MMP-2 in rat aortic SMC. MMP-2 protein and mRNA level and activity were downregulated in PKG-expressing cells as compared to PKG-deficient cells. In addition, the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased in PKG-expressing cells as compared to PKG-deficient cells. PKG-specific membrane permeable peptide inhibitor (DT-2) reverses the process. Interestingly, little or no changes of MMP-9 were observed throughout the study. Taken together our data suggest the possible role of PKG in the suppression of MMP-2.

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Gαq-phospholipase C-inositol 1,4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A2(TXA2). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA2receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA2-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor-G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA2receptors in vitro, but not Gαqcopurifying with the TXA2receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA2receptors by cyclic GMP was demonstrated from32P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA2receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA2receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both α and β forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA2receptor. These data identify TXA2receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.

Expression of Constitutively Active cGMP-Dependent Protein Kinase Prevents Glucose Stimulation of Thrombospondin 1 Expression and TGF-β Activity Shuxia Wang 1 , Xing Wu 2 , Thomas M. Lincoln 3 and Joanne E. Murphy-Ullrich 1 1 Department of Pathology, Division of Molecular and Cellular Pathology, Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 2 Department of Surgery, University of Alabama at Birmingham, Alabama 3 Department of Physiology, University of South Alabama, School of Medicine, Mobile, Alabama Address correspondence and reprint requests to Joanne E. Murphy-Ullrich, Department of Pathology, University of Alabama at Birmingham, Volker Hall 668, 1670 University Blvd., Birmingham, AL 35294-0019. E-mail: murphy{at}path.uab.edu Abstract Hyperglycemia is a crucial factor in the development of diabetic nephropathy. We previously showed that high glucose upregulates thrombospondin 1 (TSP1)-dependent transforming growth factor (TGF)-β activation by altering cGMP-dependent protein kinase (PKG) activity as a result of decreased nitric oxide signaling. In the present study, we showed that high glucose concentrations significantly reduced endogenous PKG activity. To further examine the mechanisms by which PKG regulates TSP1 expression and TSP1-dependent TGF-β activation, we generated stably transfected rat mesangial cells (RMCs) with inducible expression tetracycline-induced gene expression of the catalytic domain of PKG. After tetracycline induction, the catalytic domain of PKG is expressed as a cGMP-independent active kinase. Expression of the catalytic domain prevented high glucose-mediated increases in transcription of the TSP1 gene with no alteration in TSP1 mRNA stability. Glucose stimulation of TSP1 protein expression and TGF-β bioactivity were also downregulated. TGF-β-dependent fibronectin and type IV collagen expression under high glucose conditions were significantly reduced upon catalytic domain expression in transfected RMCs. These results show that constitutively active PKG inhibits the fibrogenic potential of high glucose through repression of TSP1-dependent TGF-β bioactivity, suggesting that gene transfer of the catalytic domain of PKG might provide a new strategy for treatment of diabetic renal fibrosis. AP-1, activator protein-1 ECM, extracellular matrix FBS, fetal bovine serum HLH, helix-loop-helix PAI-1, plasminogen activator inhibitor-1 PKG, cGMP-dependent protein kinase RMC, rat mesangial cell sTSP1, stripped thrombospondin 1 Tet-On, tetracycline-induced gene expression TetR, tetrocycline repressor TGF, transforming growth factor TSP1, thrombospondin 1 USF, upstream stimulatory factor Footnotes Accepted May 16, 2003. Received March 11, 2003. DIABETES

Nitric oxide (NO) and cyclic guanosine 3’,5’-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-Iα and PKG-Iβ, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated ‘synthetic’ VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and α-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.

HIV-infected persons entering the criminal justice system (CJS) often experience suboptimal healthcare system engagement and social instability, including homelessness. We evaluated surveys from a multisite study of 743 HIV-infected jail detainees prescribed or eligible for antiretroviral therapy (ART) to understand correlates of healthcare engagement prior to incarceration, focusing on differences by housing status. Dependent variables of healthcare engagement were: (1) having an HIV provider, (2) taking ART, and (3) being adherent (≥95% of prescribed doses) to ART during the week before incarceration. Homeless subjects, compared to their housed counterparts, were significantly less likely to be engaged in healthcare using any measure. Despite Ryan White funding availability, insurance coverage remains insufficient among those entering jails, and having health insurance was the most significant factor correlated with having an HIV provider and taking ART. Individuals interfacing with the CJS, especially those unstably housed, need innovative interventions to facilitate healthcare access and retention.

HIV and substance use are inextricably intertwined. One-sixth of people living with HIV/AIDS (PLWHA) transition through the correctional system annually. There is paucity of evidence on the impact of substance use disorders on HIV treatment engagement among jail detainees. We examined correlates of HIV treatment in the largest sample of PLWHA transitioning through jail in 10 US sites from 2007 to 2011. Cocaine, alcohol, cannabis, and heroin were the most commonly used substances. Drug use severity was negatively and independently correlated with three outcomes just before incarceration: (1) having an HIV care provider (AOR = 0.28; 95 % CI 0.09–0.89); (2) being prescribed antiretroviral therapy (AOR = 0.12; 95 % CI 0.04–0.35) and (3) high levels (>95 %) of antiretroviral medication adherence (AOR = 0.18; 95 % CI 0.05–0.62). Demographic, medical and psychiatric comorbidity, and social factors also contributed to poor outcomes. Evidence-based drug treatments that include multi-faceted interventions, including medication-assisted therapies, are urgently needed to effectively engage this vulnerable population.

Pain is highly prevalent among HIV-positive individuals, with women representing a large subset of those with pain. However, little is known about the relationship between pain and retention in HIV medical care. Among a cohort of HIV-positive women of color, we evaluated the association between pain and retention in care, as measured by missed clinic visits. The Health Resources and Services Administration’s Women of Color Initiative was a multi-site observational cohort study evaluating demonstration projects to engage HIV-positive women in medical care. From November 2010 to July 2013, 921 women were enrolled in the study across nine U.S. sites; baseline interviews collected data on socio-demographic, clinical, and risk behavior characteristics. Pain was assessed at baseline based on number of days in pain over the last 30 days and was categorized as no pain (0 days), infrequent pain (1–13 days), and frequent pain (14–30 days), with 14 days being the median. Missed visits over the one-year follow-up period, evaluated by chart abstraction, were dichotomized as ≤1 missed visit versus >1 missed visit. We conducted multivariate logistic regression to assess the association between pain at baseline and missed visits, adjusting for pertinent covariates. Among our sample (N = 862), 52.2 % of women reported no pain, 23.7 % reported infrequent pain and 24.1 % reported frequent pain. Forty-five percent had >1 missed visit during the one-year follow-up period. Overall, we did not find a significant association between pain and missed visits (aOR 2.30; 95 % CI 1.00–5.25). However, in planned stratified analyses, among women reporting current substance use at baseline, reporting frequent pain was associated with a higher odds of missed visits as compared with reporting no pain (aOR 15.14; 95 % CI 1.78–128.88). In our overall sample, pain was not significantly associated with missed visits. However, frequent pain was associated with missed visits among HIV-positive women of color who reported substance use at baseline. A better understanding of the relationship between pain and missed visits could guide efforts to improve retention in care in this population.

Vascular diseases, such as atherosclerosis and restenosis following angioplasty or transplantation, are due to abnormal vascular smooth muscle growth and gene expression. The smooth muscle cells (SMC) in response to injury lose their contractile function, become highly proliferative and synthesize and secrete extracellular matrix proteins. Similar changes in the phenotypic properties of vascular SMC occur during in vitro culture. In this report, we examined whether restoration of the expression of the major receptor protein for nitric oxide (NO) signaling in smooth muscle, the guanosine 3′:5′ cyclic monophosphate (cGMP)-dependent protein kinase (PKG), reestablished contractile function to cultured rat aortic SMC. Contractile function was monitored using the silicone polymer wrinkle assay used previously to determine contractility in cultured mesangial cells. Noncontractile rat aortic smooth muscle cells transfected with the cDNA encoding the type I isoform of PKG, but not those transfected with empty vector, formed discreet wrinkles on the substratum in response to serum indicative of contraction. Treatment of the PKG-expressing SMC with sodium nitroprusside (SNP), an NO donor, and with cGMP analogs, or with the adenylyl cyclase activator, forskolin, and with adenosine 3′:5′ cyclic monophosphate (cAMP) analogs reduced wrinkling. The expression of a major PKG substrate protein involved in smooth muscle relaxation, heat shock-related protein-20 (HSP20), was also reestablished in PKG-expressing SMC. Treatment of the PKG-expressing SMC with nitroprusside resulted in phosphorylation of HSP20. Collectively, these results indicate that PKG expression is important to establish contractility to SMC in culture.

Young persons entering US jails and youth detention facilities have high rates of sexually transmitted diseases (STDs). The Centers for Disease Control and Prevention added STD screening guidelines specific to correctional settings to the 2010 STD Treatment Guidelines. This article summarizes published evidence from 1990 to 2009 used to develop the recommendations. The literature supports routine screening of adolescents and young women (aged ≤35 years, or on the basis of local institutional prevalence data) for chlamydia and gonorrhea because of high prevalence and the subsequent risk of adverse reproductive outcomes. Chlamydia positivity among young women (aged <20 years) in juvenile detention facilities and adult facilities is more than 14%. Men in correctional settings are also at high risk for chlamydia and gonorrhea. Among boys in juvenile detention facilities, chlamydia positivity is estimated at 6.6%; among young men in adult facilities, positivity is 16.6%. Screening men (to reduce sequelae among women) should be considered based on local epidemiology and resource availability. Syphilis screening is not strongly supported in published literature because of low prevalence and is not routinely recommended; however, some screening may be warranted based on local prevalence. Although there is a great diversity in the organization of correctional facilities, implementation of screening recommendations is possible owing to improvements in test technology (urine specimens) and through integration of a standard screening protocol. Based on the high burden of disease and substantial opportunities to reach a high-risk population, correctional facilities are important venues to target efforts to control STDs.