Thrombospondin-5 and fluvastatin promote angiogenesis and are protective against endothelial cell apoptosis.

The thrombospondins (TSPs), multifunctional matricellular proteins, are known mediators of endothelial cell (EC) angiogenesis and apoptosis. TSP-1, an antiangiogenic molecule, is important in the progression of vascular disease, in part by inducing EC apoptosis. TSP-2, although less studied, also induces EC apoptosis and inhibits angiogenesis. The effects of TSP-5 are largely unexplored in ECs, but TSP-5 is believed to be protective against arterial disease. Statin drugs have been shown to have beneficial pleiotropic effects, including decreasing EC apoptosis, increasing angiogenesis, and blocking TSP signaling. We hypothesized TSP-5 will be proangiogenic and antiapoptotic, and statin pretreatment would reverse the proapoptotic and antiangiogenic phenotype of TSP-1 and TSP-2. ECs were exposed to serum-free medium, TSP-1, TSP-2, or TSP-5 with or without fluvastatin pretreatment. Quantitative real-time polymerase chain reaction was performed on 96 apoptosis and 96 angiogenesis-related genes using microfluidic card assays. Angiogenesis was measured using Matrigel assays, while apoptosis was measured by fluorescent caspase assay. TSP-5 suppressed apoptotic genes and had a mixed effect on the angiogenic genes; however, TSP-5 did not alter apoptois but was proangiogenic. Pretreatment with fluvastatin downregulated proapoptotic genes and apoptosis and upregulated proangiogenic genes and angiogenesis. Findings indicate TSP-5 and fluvastatin have a protective effect on ECs, being proangiogenic and reversing the antiangiogenic effects of TSP-1 and TSP-2. In conclusion, TSP-5 and fluvastatin may be beneficial for inducing angiogenesis in the setting of ischemia.

Fluvastatin inhibits intimal hyperplasia in wild-type but not Thbs1-null mice.

BACKGROUND: Thrombospondin-1 (TSP-1) is functionally important to intimal hyperplasia (IH) development. Statin drugs have beneficial pleiotropic effects, including reduced IH; however, the effect of statins on IH in a TSP-1-independent setting is unknown. HYPOTHESIS: Statins will be less effective in attenuating IH after vascular injury in TSP-1-null (Thbs1-/-) mice compared with wild-type (WT) mice. MATERIALS AND METHODS: Carotid artery ligation was performed on WT and Thbs1-/- mice. Each strain was divided into two groups: no statin control or standard chow containing fluvastatin (10 or 40 mg/kg/d). After 28 d, analysis included morphometric analysis and real-time quantitative reverse transcription polymerase chain reaction on the arteries and enzyme-linked immunosorbent assay on plasma (TSP-1 WT, TSP-2 WT, and Thbs1-/-). Comparisons were made by analysis of variance, with P < 0.05 considered significant. RESULTS: In no statin controls, WT mice had more IH than Thbs1-/- mice (0.46 ± 0.09 versus 0.15 ± 0.04). Fluvastatin reduced IH in the WT (0.46 ± 0.09 versus 0.23 ± 0.06), but not in Thbs1-/- groups (0.15 ± 0.04 versus 0.22 ± 0.07). No difference in IH existed between Thbs1-/- no statin controls and fluvastatin WT and Thbs1-/- groups. Statin dose did not affect IH. TSP-1 plasma levels were increased in fluvastatin WT. TSP-2 levels were decreased in fluvastatin WT and elevated in fluvastatin Thbs1-/-. Fluvastatin had no effect on tissue Thbs1 or Thbs2 gene expression. CONCLUSIONS: TSP-1 is necessary for robust IH after arterial injury. Because fluvastatin had no effect on IH in Thbs1-/-, the data suggest that the statin effect on IH may be largely TSP-1 dependent. Both statins and the presence of TSP-1 affect TSP-1 and TSP-2 plasma levels.

Thrombospondin-1 differentially regulates microRNAs in vascular smooth muscle cells.

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

Thrombospondin-1, -2 and -5 have differential effects on vascular smooth muscle cell physiology.

INTRODUCTION: The thrombospondins (TSPs) are matricellular proteins that exert multifunctional effects by binding cytokines, cell-surface receptors and other proteins. TSPs play important roles in vascular pathobiology and are all expressed in arterial lesions. The differential effects of TSP-1, -2, and -5 represent a gap in knowledge in vascular smooth muscle cell (VSMC) physiology. Our objective is to determine if structural differences of the TSPs imparted different effects on VSMC functions critical to the formation of neointimal hyperplasia. We hypothesize that TSP-1 and -2 induce similar patterns of migration, proliferation and gene expression, while the effects of TSP-5 are different. METHODS: Human aortic VSMC chemotaxis was tested for TSP-2 and TSP-5 (1-40 µg/mL), and compared to TSP-1 and serum-free media (SFM) using a modified Boyden chamber. Next, VSMCs were exposed to TSP-1, TSP-2 or TSP-5 (0.2-40 µg/mL). Proliferation was assessed by MTS assay. Finally, VSMCs were exposed to TSP-1, TSP-2, TSP-5 or SFM for 3, 6 or 24 h. Quantitative real-time PCR was performed on 96 genes using a microfluidic card. Statistical analysis was performed by ANOVA or t-test, with p < 0.05 being significant. RESULTS: TSP-1, TSP-2 and TSP-5 at 20 µg/mL all induce chemotaxis 3.1 fold compared to serum-free media. TSP-1 and TSP-2 induced proliferation 53% and 54% respectively, whereas TSP-5 did not. In the gene analysis, overall, cardiovascular system development and function is the canonical pathway most influenced by TSP treatment, and includes multiple growth factors, cytokines and proteases implicated in cellular migration, proliferation, vasculogenesis, apoptosis and inflammation pathways. CONCLUSIONS AND RELEVANCE: The results of this study indicate TSP-1, -2, and -5 play active roles in VSMC physiology and gene expression. Similarly to TSP-1, VSMC chemotaxis to TSP-2 and -5 is dose-dependent. TSP-1 and -2 induces VSMC proliferation, but TSP-5 does not, likely due conservation of N-terminal domains in TSP-1 and -2. In addition, TSP-1, -2 and -5 significantly affect VSMC gene expression; however, little overlap exists in the specific genes altered. This study further delineates TSP-1, -2 and -5's contributions to processes related to VSMC physiology.

Dyslipidemia regulates thrombospondin-1-induced vascular smooth muscle cell chemotaxis.

UNLABELLED: Dyslipidemia is a risk factor for intimal hyperplasia (IH). Key to IH is vascular smooth muscle cell (VSMC) migration. Thrombospondin-1 (TSP-1) is a matricellular protein that stimulates VSMC migration. HYPOTHESIS: HDL will inhibit and LDL will augment TSP-1-induced VSMC chemotaxis. VSMC chemotaxis will be inhibited by the HDL moiety, S1P, through the S1PR1 receptor, and augmented by the LDL component, LPA, through the LPAR1 receptor. The goal of this study was to determine the effect of HDL and LDL and their receptors on TSP-1-induced VSMC chemotaxis. For VSMC chemotaxis to TSP-1 cells received the following pretreatments: low (25 µg/ml) or optimal (75 µg/ml) concentration of HDL, S1P, optimal (75 µg/ml) or high (175 µg/ml) concentration of LDL, or LPA. For the receptor studies, VSMCs were transfected with siRNA to S1PR1, S1PR3, LPAR1, LPAR2, LPAR3, or a S1PR2 receptor antagonist. The TSP-1-induced chemotaxis results were (1) HDL (25 µg/ml) or LDL (75 µg/ml) exhibited no effect on chemotaxis; (2) HDL (75 µg/ml) inhibited chemotaxis by 50.9 ± 8 % and S1P by 43.4 ± 11.6 %; (3) LDL (175 µg/ml) augmented chemotaxis by 30 ± 10.4 % and LPA by 25.6 ± 12.3 %; (4) S1PR1 and S1PR3 knockdown and S1PR2 antagonist-treated cells augmented chemotaxis; and (5) LPAR1 and LPAR2 knockdown inhibited and LPAR3 knockdown had no effect on chemotaxis. In conclusion, HDL/S1P inhibits, while LDL/LPA stimulates TSP-1-induced VSMC chemotaxis. The HDL/S1P effect is mediated by the S1PR1-3 receptors. The LDL/LPA effects are mediated by the LPAR1 and LPAR2 receptors, but not LPAR3. Therefore, lipids have significant effects on TSP-1-induced VSMC chemotaxis.

Thrombospondin-1-induced smooth muscle cell chemotaxis and proliferation are dependent on transforming growth factor-ß2 and hyaluronic acid synthase.

Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-ß2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-ß2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-ß2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-ß2 inhibition and unaffected by HAS inhibition. TGF-ß2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-ß2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-ß2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-ß2 and HAS.

The role of hyaluronic acid in atherosclerosis and intimal hyperplasia.

Atherosclerosis is a chronic inflammatory condition of the blood vessel wall that can lead to arterial narrowing and subsequent vascular compromise. Although there are a variety of open and endovascular procedures used to alleviate the obstructions caused by atherosclerotic plaque, blood vessel instrumentation itself can lead to renarrowing of the vessel lumen through intimal hyperplasia, wound contracture, or a combination of the two. While the cell types involved in both atherosclerosis and vessel renarrowing after surgical intervention are largely characterized, current research has shown that components of the extracellular matrix are also important in the pathogenesis of the aforementioned processes. One such component is hyaluronic acid (HA). The objective of this review, therefore, is to examine the involvement of HA in these pathologic processes. Literature on the structure and function of HA was reviewed, with particular attention given to the role of HA in the processes of atherogenesis, intimal hyperplasia, and wound contracture after blood vessel instrumentation. HA interacts with vascular smooth muscle cells (VSMCs), endothelial cells (ECs), and platelets to promote atherogenesis. In particular, VSMCs manufacture large amounts of HA that form "cable-like" structures important for leukocyte adhesion and rolling. Additionally, transmigration of leukocytes across the EC layer is mediated by HA. Platelets cleave large molecules of HA into fragments that up-regulate leukocyte production of chemokines and cytokines. HA also has a role in both intimal hyperplasia and wound contracture, the two processes most responsible for vessel renarrowing after vascular instrumentation. HA has a complex, and sometimes conflicting, role in the pathologic processes of atherogenesis and vessel wall renarrowing after surgical intervention.

Lovastatin inhibits thrombospondin-1-induced smooth muscle cell chemotaxis.

BACKGROUND: Thrombospondin-1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration and is important in the development of intimal hyperplasia. HMG-CoA reductase inhibitors, such as lovastatin, reduce the incidence of vascular restenosis after angioplasty by both cholesterol lowering and pleiotropic effects. Inhibition of the mevalonate pathway is largely responsible for these pleiotropic properties. This inhibition prevents isoprenylation of the small G proteins, Rho and Ras, by geranylgeranyl and farnesyl pyrophosphate, respectively. Isoprenylation is required for Ras and Rho activation, which is relevant for cell migration. HYPOTHESIS: Lovastatin inhibits TSP-1-induced VSMC chemotaxis by inhibiting small G proteins via the mevalonate pathway. METHODS: Chemotaxis was assessed using a modified Boyden chamber. Quiescent VSMCs were pretreated with serum free media (SFM), lovastatin with or without mevalonate farnesyl (FTI), geranylgeranyl transferase inhibitors (GGTI), farnesyl transferase inhibitor (FPT), or the Rho kinase inhibitor (Y-27632). Chemoattractants were SFM or TSP-1. Comparisons were made by ANOVA followed by post-hoc testing (P<0.05). The effect of lovastatin on Ras activation was evaluated using cells pretreated with SFM or lovastatin, with or without mevalonate prior to TSP-1 exposure. Western blot for Ras activation was performed. RESULTS: Lovastatin dose-dependently inhibited TSP-1-induced chemotaxis, which was reversed by mevalonate. Mevalonate did not induce chemotaxis independently. FTI and FPT, but not GGTI or Y-27632, inhibited TSP-1-induced Ras activation and TSP-1-induced chemotaxis. Lovastatin inhibition of Ras activation was reversed with mevalonate. CONCLUSION: Ras, not Rho, is relevant for TSP-1-induced VSMC chemotaxis. These data suggest that lovastatin suppresses TSP-1-induced chemotaxis by inhibition of Ras.

A clinically applicable porcine model of septic and ischemia/reperfusion-induced shock and multiple organ injury.

BACKGROUND: Although many sepsis treatments have shown efficacy in acute animal models, at present only activated protein C is effective in humans. The likely reason for this discrepancy is that most of the animal models used for preclinical testing do not accurately replicate the complex pathogenesis of human sepsis. Our objective in this study was to develop a clinically applicable model of severe sepsis and gut ischemia/reperfusion (I/R) that would cause multiple organ injury over a period of 48 h. MATERIALS AND METHODS: Anesthetized, instrumented, and ventilated pigs were subjected to a "two-hit" injury by placement of a fecal clot through a laparotomy and by clamping the superior mesenteric artery (SMA) for 30 min. The animals were monitored for 48 h. Wide spectrum antibiotics and intravenous fluids were given to maintain hemodynamic status. FiO(2) was increased in response to oxygen desaturation. Twelve hours following injury, a drain was placed in the laparotomy wound. Extensive hemodynamic, lung, kidney, liver, and renal function measurements and serial measurements of arterial and mixed venous blood gases were made. Bladder pressure was measured as a surrogate for intra-peritoneal pressure to identify the development of the abdominal compartment syndrome (ACS). Plasma and peritoneal ascites cytokine concentration were measured at regular intervals. Tissues were harvested and fixed at necropsy for detailed morphometric analysis. RESULTS: Polymicrobial sepsis developed in all animals. There was a progressive deterioration of organ function over the 48 h. The lung, kidney, liver, and intestine all demonstrated clinical and histopathologic injury. Acute lung injury (ALI) and ACS developed by consensus definitions. Increases in multiple cytokines in serum and peritoneal fluid paralleled the dysfunction found in major organs. CONCLUSION: This animal model of Sepsis+I/R replicates the systemic inflammation and dysfunction of the major organ systems that is typically seen in human sepsis and trauma patients. The model should be useful in deciphering the complex pathophysiology of septic shock as it transitions to end-organ injury thus allowing sophisticated preclinical studies on potential treatments.

Thrombospondin-1: a proatherosclerotic protein augmented by hyperglycemia.

OBJECTIVE: Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces vascular smooth muscle (VSMC) migration and proliferation in areas of vascular injury and is also up-regulated in VSMCs exposed to hyperglycemia. This study tested the hypothesis that hyperglycemia amplifies the expression of genes induced by TSP-1 in VSMCs. METHODS: Human aortic VSMCs were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were used between passages three and five. VSMCs were preincubated in DMEM containing 0.2% FBS with 5 mM glucose (normoglycemia), 25 mM glucose (hyperglycemia), 25 mM mannose (osmotic control), TSP-1 (20 microg/mL), 25 mM glucose + TSP-1 (20 microg/mL), or 25 mM mannose + TSP-1 (20 microg/mL). Total RNA was extracted. Microarray analysis was performed and analyzed by analysis of variance. P < .05 was considered significant. Quantitative real-time polymerase chain reaction (rtPCR) was used to confirm selected up-regulated genes. RESULTS: Microarray analysis revealed: (1) hyperglycemia altered 30 genes; (2) TSP-1 altered 212 genes, of which 8 were altered similarly to VSMCs exposed to 25 mM glucose; (3) TSP-1 up-regulated 10 genes associated with atherosclerosis and 4 others with diabetic vascular disease; (4) hyperglycemia combined with TSP-1 altered expression of 2822 genes. The three genes most up-regulated by TSP-1 in a normoglycemic environment were uridine 5'-diphosphoglucose (UDP-glucose) dehydrogenase (UGDH, 127%), transforming growth factor beta-2 (TGFbeta2, 116%), and hyaluronan synthase 2 (HAS2, 113%). Further, TSP-1 altered the expression of genes in 13 canonical pathways; however, when combined with hyperglycemia, 53 canonical pathways were affected. CONCLUSION: Quantitative rtPCR confirmed that genes in several of these pathways for TSP-1 and hyperglycemia combined with TSP-1 were up-regulated. These findings suggest that TSP-1 may be germane to the progression of atherosclerosis and may have a large effect with concurrent hyperglycemia.

Inhibition of heat shock protein 90 attenuates post­angioplasty intimal hyperplasia.

Intimal hyperplasia (IH) is a pathologic process that leads to restenosis after treatment for peripheral arterial disease. Heat shock protein 90 (HSP90) is a molecular chaperone that regulates protein maturation. Activation of HSP90 results in increased cell migration and proliferation. 17­N­allylamino­17­demethoxygeldanamycin (17­AAG) and 17­dimethylaminoethylamino­17­demethoxygeldanamycin (17­DMAG) are low toxicity Food and Drug Association approved HSP90 inhibitors. The current study hypothesized that HSP90 inhibition was predicted to reduce vascular smooth muscle cell (VSMC) migration and proliferation. In addition, localized HSP90 inhibition may inhibit post­angioplasty IH formation. For proliferation, VSMCs were treated with serum­free media (SFM), 17­DMAG or 17­AAG. The selected proliferative agents were SFM, platelet derived growth factor (PDGF) or fibronectin. After three days, proliferation was measured. For migration, VSMCs were treated with SFM, 17­AAG or 17­DMAG with SFM, PDGF or fibronectin as chemoattractants. Balloon injury to the carotid artery was performed in rats. The groups included in the present study were the control, saline control, 17­DMAG in 20% pluronic gel delivered topically to the adventitia or intraluminal delivery of 17­DMAG. After 14 days, arteries were fixed and sectioned for morphometric analysis. Data was analyzed using ANOVA or a student's t­test. P<0.05 was considered to indicate a statistically significant difference. The results revealed that 17­AAG and 17­DMAG had no effect on cell viability. PDGF and fibronectin also increased VSMC proliferation and migration. Furthermore, both 17­AAG and 17­DMAG decreased cell migration and proliferation in all agonists. Topical adventitial treatment with 17­DMAG after balloon arterial injury reduced IH. HSP90 inhibitors suppressed VSMC proliferation and migration without affecting cell viability. Topical treatment with a HSP90 inhibitor (DMAG) decreased IH formation after arterial injury. It was concluded that 17­DMAG may be utilized as an effective therapy to prevent restenosis after revascularization.

Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury.

INTRODUCTION: Oral statins reduce intimal hyperplasia (IH) after arterial injury by only ∼25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid-tagged polysialic acid-polycaprolactone micelles) on vascular smooth muscle cell (VSMC) migration, VSMC proliferation and intimal hyperplasia. We hypothesized both free and micelle containing simvastatin would inhibit VSMC chemotaxis and proliferation, and local statin treatment would be more effective than oral in reducing IH in rats following carotid balloon injury. METHODS: VSMCs pretreated with free simvastatin (20 minutes or 20 hours) or simvastatin-loaded micelles underwent chemotaxis and proliferation to platelet-derived growth factor. Next, rats that underwent balloon injury of the common carotid artery received statin therapy-intraluminal simvastatin-loaded micelles prior to injury, periadventitial pluronic gel following injury, or combinations of gel, micelle, and oral simvastatin. After 14 days, morphometric analysis determined the -intimal to medial ratio. Findings were compared to controls receiving oral simvastatin or no statin therapy. Statistical analysis was by analysis of variance for the in vitro experiments and a factorial general linear model for the in vivo experiments. RESULTS: The simvastatin-loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively); however, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03). Addition of oral statins or combining both local therapies did not provide additional benefit. Micelles were the single greatest contributing factor in IH attenuation. CONCLUSIONS: Intraluminally or topically delivered statins reduced IH. The efficacy of single-dose, locally delivered statin alone may lead to novel treatments to prevent IH. The different routes of administration may allow for treatment during endovascular procedures, without the need for systemic therapy.

An interleukin-6-neutralizing antibody prevents cyclosporine-induced nephrotoxicity in mice.

INTRODUCTION: Chronic use of cyclosporine A (CyA) induces nephrotoxicity primarily due to endothelial dysfunction. In our previous studies, potential mechanisms were identified in vitro and implicated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin-6 (IL-6) as key components in causing endothelial dysfunction. In this study, we tested the hypothesis that NADPH oxidase activity and IL-6 are key components in renal damage in an in vivo model. METHODS: Male mice C57B/6 mice from Jackson Laboratory (Bar Harbor, ME) at 6-8 wks were subjected to a low-salt diet throughout the trial. After 1 week on a low-salt diet, the mice were injected daily with treatments in 50 muL vehicle composed of 75% cremaphor (Sigma, St. Louis, MO) and ethanol for 5 wks. A vehicle-alone group was also set aside. Mice were weighed and 25 mg/kg/day cyclosporine (Novartis Pharma, St. Louis, MO) was injected daily. Apocynin (Calbiochem, Gibbstown, NJ) 20 mg/kg were injected either alone or concomitantly with CyA. Another group of mice were administered IL-6 antibody (Cat no. MAB406; R&D Systems, Minneapolis, MN) at 2 mug/day along with CyA. The kidneys were removed en bloc immediately and submitted in formalin for paraffin sections. Trichrome stains were performed. Slides were blinded and 10 photographs of cortical areas per treatment group were taken, which covered an estimate of 10% surface area in random fashion. Areas of renal damage, which were determined by tubular necrosis, were identified and quantified by amount of necrosis per photograph. Each photograph was divided into 10 blocks, and the number of blocks that contained necrotic tubules per photo was recorded. RESULTS: The two control mice (low salt only) had no damage. The four vehicle mice had trace amounts of tubular necrosis. CyA treatment group demonstrated the highest amount of damage (29/70; 41%). CyA with apocynin, a specific NADPH oxidase inhibitor, was found to have 36% (22/60) damage, whereas the CyA with IL-6 antibody only was observed to have 15% (6/40) damage. Comparing imaging analysis, there was no difference between mice treated with CyA alone and with CyA with apocynin. However, the amount of damage in mice treated with CyA and IL-6 antibody was found to be significantly lower than both CyA and CyA with apocynin. CONCLUSIONS: CyA action as a calcineurin inhibitor has allowed prolongation of kidney transplants, but its chronic use has led to devastating consequences such as allograft nephropathy. Previously, we have identified potential mechanisms of CyA-induced endothelial dysfunction in vitro. The current study identifies increased IL-6 expression as a mechanism by which CyA induces renal damage and that the use of an IL-6-neutralizing antibody may be useful in reducing CyA-induced renal damage.

Cyclosporine inhibition of angiogenesis involves the transcription factor HESR1.

PURPOSE: Angiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506. METHODS: HAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction. RESULTS: At a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment. CONCLUSION: CyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

Nicotinamide adenine dinucleotide phosphate oxidase and diabetes: vascular implications.

Vascular disease associated with diabetes mellitus is a major cause of morbidity and mortality and is increasing in the United States. It is now recognized that oxidative stress plays a substantial role in the underlying vascular pathology of several diseases, including hypertension and diabetes. In diabetes, there is an increase in the steady state levels of reactive oxygen species. One of the primary generators of reactive oxygen species is nicotinamide adenine dinucleotide phosphate oxidase. Studies have indicated that inhibition of this system is associated with vascular benefits in diabetes. Therefore, there may be a role for therapies directed at nicotinamide adenine dinucleotide phosphate oxidase in this disease. This review will examine the structure, activation, potential role in vascular disease, and benefits of inhibition of nicotinamide adenine dinucleotide phosphate oxidase.

Dyslipidemia Part 1--Review of Lipid Metabolism and Vascular Cell Physiology.

Dyslipidemia, more specifically, high-serum low-density lipoproteins and low-serum high-density lipoproteins, are known risk factors for cardiovascular disease. The current clinical treatment of dyslipidemia represents the outcome of a large body of fundamental basic science research on lipids, lipid metabolism, and the effects of different lipids on cellular components of the artery, inflammatory cells, and platelets. In general, lower density lipids activate intracellular pathways to increase local and systemic inflammation, monocyte adhesion, endothelial cell dysfunction and apoptosis, and smooth muscle cell proliferation, resulting in foam cell formation and genesis of atherosclerotic plaque. In contrast, higher density lipids prevent or attenuate atherosclerosis. This article is part 1 of a 2-part review, with part 1 focusing on lipid metabolism and the downstream effects of lipids on the development of atherosclerosis, and part 2 on the clinical treatment of dyslipidemia and the role of these drugs for patients with arterial disease exclusive of the coronary arteries.

Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.

In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs.

Effects of a 20-HETE antagonist and agonists on cerebral vascular tone.

This study examined the effects of a 20-hydroxyeicosatetraenoic acid (20-HETE) antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (WIT002) and two agonists, 4-amino-N-(20-hydroxy-eicosa-5(Z),14(Z)-dienoyl) benzenesulfonamide (ABSA) and 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (WIT003), on the diameter of rat middle cerebral arteries in vitro and on cerebral blood flow in vivo. WIT003, ABSA and 20-HETE all had a similar effect to reduce the diameter of the middle cerebral artery by 26%. WIT003 and 20-HETE both increased intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells isolated from the middle cerebral artery. In contrast, WIT002 had no effect on the basal diameter of the middle cerebral artery but it attenuated the vasoconstrictor responses and the rise in [Ca2+]i in vascular smooth muscle cells following administration of 20-HETE and 5-hydroxytryptamine (5-HT). WIT003 partially restored the vasoconstrictor response to 5-HT in the middle cerebral artery after administration of an inhibitor of the endogenous synthesis of 20-HETE. Infusion of the 20-HETE agonists, WIT003 and ABSA, into cisterna magna of rats reduced baseline cerebral blood flow by 20%, whereas administration of the 20-HETE antagonist, WIT002, had no effect. Intracisternal injection of WIT002 attenuated the fall in cerebral blood flow following injection of blood into the cisterna magna, whereas administration of the 20-HETE agonist, ABSA, potentiated this response. These findings indicate that the 20-HETE agonists, WIT003 and ABSA, increase cerebral vascular tone both in vivo and in vitro and suggest blocking the vasoconstrictor actions of 20-HETE may be useful to prevent the acute fall in cerebral blood flow following subarachnoid hemorrhage.

Statins and nitric oxide donors affect thrombospondin 1-induced chemotaxis.

BACKGROUND: Thrombospondin 1 (TSP-1) induces vascular smooth muscle cell (VSMC) migration and intimal hyperplasia. Statins and nitric oxide (NO) donors decrease intimal hyperplasia. We previously showed that statins (long-term exposure) and NO donors inhibit TSP-1-induced VSMC chemotaxis. HYPOTHESES: (1) Pretreatment with short-term statin will inhibit TSP-1-induced VSMC chemotaxis and (2) NO donors will enhance statin inhibition of TSP-1-induced or platelet-derived growth factor (PDGF)-induced VSMC chemotaxis. METHODS: We examined these treatment effects on TSP-1-induced VSMC chemotaxis: (1) long-term (20 hours) versus short-term (20 minutes) pravastatin, (2) diethylenetriamine NONOate (DETA/NO) or S-nitroso-N-acetylpenicillamine (SNAP) in combination with pravastatin, and (3) comparison of TSP-1 to PDGF as a chemoattractant. RESULTS: Pravastatin (long term or short term) inhibited TSP-1-induced chemotaxis. Diethylenetriamine NONOate and SNAP impeded statin inhibition of TSP-1-induced chemotaxis. Platelet-derived growth factor and TSP-1 had opposite effects on DETA/NO-pravastatin treatment. CONCLUSION: Short-term statin pretreatment inhibited TSP-1-induced VSMC chemotaxis, suggesting a pleiotropic effect. High-dose NO reversed statin inhibition of TSP-1-induced chemotaxis, suggesting NO and statin combination therapies warrant further study.