David J. O’Regan

Human cardiac fibroblasts express ICAM-1, E-selectin and CXC chemokines in response to proinflammatory cytokine stimulation

Neutrophil attraction and adhesion to endothelial cells occurs via well defined mechanisms, yet the ability of other cell types to express neutrophil-binding adhesion molecules is not well studied. Cardiac fibroblasts (CF) are a key cell type involved in repair of the infarcted myocardium, a scenario in which neutrophil recruitment is perceived to be detrimental. Here we determined the effects of proinflammatory cytokines on expression of neutrophil-binding adhesion molecules and neutrophil-attracting chemokines in CF cultured from multiple patients, and explored the underlying regulatory mechanisms. An adhesion molecule focused RT-PCR array identified 5 transcripts that were increased markedly in human CF treated with the proinflammatory cytokine interleukin-1 (IL-1, 10 ng/ml, 6 h); including intercellular cell adhesion molecule (ICAM-1) and E-selectin. Real-time RT-PCR verified the array data and immunoblotting confirmed cytokine-induced ICAM-1 and E-selectin protein expression. Treatment with a panel of pharmacological inhibitors identified the NF-κB pathway as mediating IL-1-induced ICAM-1 and E-selectin mRNA and protein expression. Additionally, E-selectin expression in human CF was markedly potentiated by the JNK inhibitor SP600125, but this was not observed when a more selective inhibitor ((L)-JNKI-1) was used, or in human vascular endothelial cells. IL-1 also stimulated CF to secrete the neutrophil chemoattractant CXCL8 via a p38- and NF-κB-dependent mechanism, as well as inducing CXCL1, CXCL2 and CXCL5 mRNA expression. In conclusion, human CF express neutrophil-binding adhesion molecules and neutrophil chemoattractants in response to proinflammatory cytokines suggesting that, in addition to EC, CF may play an important role in regulating neutrophil recruitment into the infarcted myocardium.

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01–10 μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1 μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ-INDEPENDENT EFFECTS OF THIAZOLIDINEDIONES ON HUMAN CARDIAC MYOFIBROBLAST FUNCTION

1 Thiazolidinediones (TZDs) are peroxisome proliferator‐activated receptor (PPAR) γ agonists that are used to lower insulin resistance in Type 2 diabetic patients. Although TZDs exhibit beneficial effects on the vasculature, their effects on the heart are less clear and are the subject of current clinical debate. Thiazolidinediones have been reported to reduce adverse myocardial remodelling, a pathology in which cardiac myofibroblasts (CMF) are pivotal. 2 The aim of the present study was to investigate whether TZDs modulate specific human CMF functions of importance to the myocardial remodelling process and to determine whether any of these effects were mediated via PPARγ activation. 3 Immunoblotting of cultured human CMF homogenates revealed strong expression of PPARγ (approximately 50 kDa). Three different TZDs (ciglitazone, rosiglitazone and troglitazone) and the endogenous PPARγ ligand 15‐deoxy‐δ12,14‐prostaglandin J2 (15d‐PGJ2) inhibited CMF proliferation (cell number and expression of proliferating cell nuclear antigen) in a concentration‐dependent manner (range 0.1–10 µmol/L) with similar potencies. This antiproliferative effect of TZDs was not reversed by the PPARγ antagonists GW9662 or T0070907 (10–25 µmol/L). None of the TZDs or 15d‐PGJ2 affected cell migration or invasion (Boyden chamber assays without or with Matrigel barrier), matrix metalloproteinase‐2 or ‐9 secretion (gelatin zymography) or the actin cytoskeleton (rhodamine/phalloidin fluorescent confocal microscopy). 4 In conclusion, TZDs reduce human CMF proliferation via a PPARγ‐independent mechanism. Although TZDs do not inhibit CMF invasion, their antiproliferative activity may contribute to the ability of this class of drugs to modulate adverse myocardial remodelling.

Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: Possible role of differential RhoA activity

BACKGROUND/PURPOSE Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. METHODS/MATERIALS SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. RESULTS IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P<0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P<0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P<0.05 and 43% reduction in activity, P<0.01). CONCLUSIONS IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients. SUMMARY The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.

p38 MAPK alpha mediates cytokine-induced IL-6 and MMP-3 expression in human cardiac fibroblasts

Highlights ► The p38 MAPK signaling pathway regulates cardiac remodeling. ► Human cardiac fibroblasts express the α, γ and δ subtypes of p38 MAPK. ► IL-1 selectively stimulates p38α and p38γ activation. ► p38α is important for IL-1-induced IL-6 and MMP-3 expression in cardiac fibroblasts.

Investigating inherent functional differences between human cardiac fibroblasts cultured from nondiabetic and Type 2 diabetic donors

INTRODUCTION Type 2 diabetes mellitus (T2DM) promotes adverse myocardial remodeling and increased risk of heart failure; effects that can occur independently of hypertension or coronary artery disease. As cardiac fibroblasts (CFs) are key effectors of myocardial remodeling, we investigated whether inherent phenotypic differences exist in CF derived from T2DM donors compared with cells from nondiabetic (ND) donors. METHODS Cell morphology (cell area), proliferation (cell counting over 7-day period), insulin signaling [phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) Western blotting], and mRNA expression of key remodeling genes [real-time reverse transcription-polymerase chain reaction (RT-PCR)] were compared in CF cultured from atrial tissue from 14 ND and 12 T2DM donors undergoing elective coronary artery bypass surgery. RESULTS The major finding was that Type I collagen (COL1A1) mRNA levels were significantly elevated by twofold in cells derived from T2DM donors compared with those from ND donors; changes reflected at the protein level. T2DM cells had similar proliferation rates but a greater variation in cell size and a trend towards increased cell area compared with ND cells. Insulin-induced Akt and ERK phosphorylation were similar in the two cohorts of cells. CONCLUSION CF from T2DM individuals possess an inherent profibrotic phenotype that may help to explain the augmented cardiac fibrosis observed in diabetic patients. MINI SUMMARY We investigated whether inherent phenotypic differences exist between CF cultured from donors with or without Type 2 diabetes. Cell morphology, proliferation, insulin signaling, and gene expression were compared between multiple cell populations. The major finding was that Type I collagen levels were elevated in fibroblasts from diabetic donors, which may help explain the augmented cardiac fibrosis observed with diabetes.

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor

Objective—Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca2+) entry. Here, we sought understanding of a Synta66-resistant Ca2+ entry pathway activated by VEGF. Approach and Results—Measurement of intracellular Ca2+ in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca2+ entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca2+ entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase C&ggr;1, Ca2+ release, cytosolic group IV phospholipase A2&agr;, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4–synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. Conclusions—VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca2+ entry and downstream endothelial cell remodeling.

The −1562C/T MMP-9 promoter polymorphism does not predict MMP-9 expression levels or invasive capacity in saphenous vein smooth muscle cells cultured from different patients

Matrix metalloproteinase-9 (MMP-9) is an important regulator of vascular smooth muscle cell (SMC) invasion and proliferation. The T allele of the -1562C/T MMP-9 promoter polymorphism reportedly confers increased MMP-9 promoter activity, plasma MMP-9 levels and susceptibility to vascular pathologies. The aim of this study was to determine whether the MMP-9 -1562C/T polymorphism directly influences endogenous MMP-9 expression levels in saphenous vein (SV) SMC cultured from patients with different genotypes. Genotyping of 408 patients revealed -1562C/T genotype frequencies of 73.3% CC, 25.0% CT and 1.7% TT. Using a standardized, controlled protocol we investigated the effects of phorbol ester (TPA) and a physiological stimulus (PDGF+IL-1) on MMP-9 expression in cultured SV-SMC from 15 CC, 15 CT and 3 TT patients, and on PDGF+IL-1-induced SV-SMC invasion (Boyden chamber with Matrigel barrier). A strong correlation between MMP-9 mRNA levels (real-time RT-PCR) and MMP-9 protein secretion (gelatin zymography) was observed. However, no significant differences were observed in MMP-9 expression levels, or in SV-SMC invasion, between cells with different -1562C/T genotypes. Moreover, MMP-9 promoter activity of the C and T variants was similar. Our data challenge the functional nature of the -1562C/T polymorphism and its capacity to modulate MMP-9 expression levels and SV-SMC invasion, and hence susceptibility to vascular pathologies in vivo.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVβ3 and RhoA/ROCK-mediated mechanisms

Graphical abstract

MMP-3 (5A/6A) Polymorphism Does Not Influence Human Smooth Muscle Cell Invasion

BACKGROUND Stromelysin (MMP-3) is an important regulator of vascular smooth muscle cell (SMC) invasion, a key contributor to saphenous vein (SV) bypass graft failure. The 5A allele of the common -1612 MMP-3 5A/6A promoter polymorphism reportedly confers increased promoter activity, MMP-3 tissue expression, and susceptibility to a number of vascular pathologies. The aim of this study was to determine whether the MMP-3 5A/6A polymorphism directly influences endogenous MMP-3 expression levels and, consequently, cell invasion, in SV-derived SMC cultured from patients with different genotypes. MATERIAL AND METHODS Genotyping of 226 patients revealed -1612 MMP-3 5A/6A genotype frequencies of 20.8% 5A/5A, 52.7% 5A/6A, and 26.5% 6A/6A. Using a standardized, controlled protocol, we investigated cytokine- and growth factor-induced MMP-3 expression (real-time polymerase chain reaction [RT-PCR], ELISA) and SV-SMC invasion (Boyden chamber with Matrigel barrier) using cultured SV-SMC from patients with different MMP-3 genotypes. RESULTS Despite observing a strong correlation between MMP-3 mRNA levels and MMP-3 protein secretion, no significant differences were apparent in MMP-3 expression levels or cell invasion between cells with different MMP-3 5A/6A genotypes. CONCLUSIONS Our data suggest that the MMP-3 5A/6A promoter polymorphism in isolation does not influence levels of MMP-3 secretion or cellular invasion in human SV-SMC.