Thrombospondins Differentially Regulate Proteins Involved in Arterial Remodeling.

Thrombospondins (TSPs) are matricellular glycoproteins expressed in response to vascular injury. TSP-1 and TSP-2 are promotors of arterial remodeling while TSP-5 is believed to be protective. The current study assessed the differential effect of TSPs on protein expression in vascular smooth muscle cells (VSMCs). We hypothesized that TSP-1, TSP-2 and TSP-5 would regulate VSMC proteins involved in arterial remodeling. Human VSMCs were exposed to TSP-1, -2, -5 or serum free media (24 hours). Cell lysates were used to assess the targets TSP-1, TSP-2, TSP-5 and CD44), while the culture media was used to detect TGF-ß1, PDGF-BB, ANGPTL-4 and IL-8. Statistical analysis was performed by t-test and p< 0.05 was considered significant. All TSPs increased their own expression and TSP-5 increased TSP-2. TSP-1 and TSP-2 increased production of ANGPTL-4 and PDGF-BB, while TSP-5 only increased ANGPTL-4. TSP-1 increased exclusively TGF-ß1 and CD44 production. TSP-2 increased TSP-1 expression. All TSPs decreased IL-8. The findings suggest that TSP-1 and TSP-2 may promote vascular remodeling, in part, by increasing ANGPTL-4, PDGF-BB and their own expression. TSP-5 did not upregulate the inflammatory mediators TSP-1, PDGF-BB or TGF-ß1, but upregulated its own expression, which could be a protective mechanism against the response to vascular injury.

Vascular smooth muscle cell migration: current research and clinical implications.

Atherosclerosis and intimal hyperplasia are major causes of morbidity and mortality. These processes develop secondary to endothelial injury due to multiple stimuli, including smoking, diabetes mellitus, hypertension, and hyperlipidemia. Once this injury occurs, an essential element in the development of both these processes is vascular smooth muscle cell (VSMC) migration. Understanding the mechanisms involved in VSMC migration and ultimately the development of strategies by which this process can be inhibited, has been a major focus of research. The authors present a review of the extracellular proteins (growth factors, extracellular matrix components, and cell surface receptors) and intracellular signaling pathways involved in VSMC migration, as well as potential therapeutic approaches to inhibit this process.

Peripheral vascular disease intervention in patients with end-stage renal disease: few complications in those treated with peritoneal dialysis.

BACKGROUND: We assessed the results of peripheral vascular surgery in patients with end-stage renal disease (ESRD) who were being treated with peritoneal dialysis. METHODS: Sixty-seven ESRD patients on peritoneal dialysis who had peripheral vascular surgery were assessed retrospectively for preoperative risk factors, primary and secondary patency rates, and mortalitv. The study group had 48 proximal femoral-popliteal bypasses, 12 distal femoral-popliteal bypasses, and 7 distal femoral-tibial and/or peroneal revascularizations. RESULTS: Among 67 peritoneal dialysis patients, 15 deaths (22%) occurred over 68 months (mean, 14 months). CONCLUSION: Patients on peritoneal dialysis had adequate patency rates and length of survival after peripheral vascular surgery when maintained on peritoneal dialysis.

Calciphylaxis: a poor prognostic indicator for limb salvage.

INTRODUCTION: Calciphylaxis (calcific uremic arteriolopathy) is a rare complication of end-stage renal disease in patients with secondary hyperparathyroidism. Subcutaneous skin nodules and extensive tissue necrosis with vascular calcification characterize this process. DESIGN AND SETTING: The design of the study included case series and literature review. The study was set in a tertiary care hospital. PATIENTS: Four patients presented over an 8-month period with extensive tissue loss and the subsequent diagnosis of calciphylaxis. Two of these patients were revascularized. One underwent primary amputation, and the final patient died before revascularization. The mortality rate for this group was 75%. All patients had significant vascular complications outside the area of initial presentation. CONCLUSIONS: These individuals represent a subset of patients who may not benefit from revascularization or may require extensive regulation of divalent metabolism before consideration for revascularization.

The C-terminal domain of thrombospondin-1 induces vascular smooth muscle cell chemotaxis.

OBJECTIVE: Thrombospondin-1 (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-1 has six domains of repeating homologous amino acid sequences: N-terminal, procollagen homology, type 1 repeat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The purpose of this experiment was to determine which domains of TSP-1 induce VSMC chemotaxis. METHODS: A modified Boyden Chamber chemotaxis assay was used to assess VSMC migration. Serum-free medium, TSP-1, or each of the fusion proteins (10 and 20 microg/mL) synthesized for the different domains were placed in the bottom wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top wells. After 4 hours at 37 degrees C, migrated VSMCs were recorded as cells per five fields (400x) and analyzed with the paired t test. To verify the fusion protein data, we performed chemotaxis assays with antibodies to each of the domains (25 microg/mL) combined with TSP-1 (20 microg/mL) in the bottom wells and VSMCs in the top wells. RESULTS: The COOH domain significantly stimulated VSMC chemotaxis (P = <.001). To a lesser extent, the N-terminal and T3 domains also induced chemotaxis (P <.05). However, only the anti-COOH antibody (C6.7) and the anti-integrin-associated protein portion of COOH antibody (D4.6) significantly inhibited TSP-1-induced VSMC chemotaxis (by 85% and 92%, respectively). CONCLUSIONS: These results implicate the COOH domain as the portion of the TSP-1 molecule primarily responsible for VSMC chemotaxis. This experiment suggests that future strategies in the prevention of VSMC migration, an initial step in the development of vascular lesions, may involve selective inhibition of the COOH domain of TSP-1.

Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress.

The p38/mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAP kinase 2)/heat shock protein (HSP)25/27 pathway is thought to play a critical role in actin dynamics. In the present study, we examined whether p38 was involved in the morphological changes seen in endothelial cells (EC) exposed to shear stress. Cultured bovine aortic EC were subjected to 14 dyn/cm(2) laminar steady shear stress. Peak activation of p38, MAPKAP kinase 2, and HSP25 were sixfold at 5 min, sixfold at 5 min, and threefold at 30 min compared with static control, respectively. SB-203580 (1 microM), a specific inhibitor of p38, abolished the activation of MAPKAP kinase 2 and HSP25 as well as EC elongation and alignment in the direction of flow elicited by shear stress. The mean orientation angle of cells subjected to shear without SB-203580, with SB-203580, or static control were 17, 50, and 43 degrees, respectively (P < 0. 05). EC transfected with the dominant negative mutant of p38-alpha aligned randomly with no stress fiber formation despite exposure to shear stress. These data suggests that the pathway of p38/MAPKAP kinase 2/HSP25/27 is activated in response to shear stress, and this pathway plays an important role in morphological changes induced by shear stress.

Role of mitogen-activated protein kinases in pulmonary endothelial cells exposed to cyclic strain.

The aim of this study was to examine the role of mitogen-activated protein kinases (MAPKs) activation in bovine pulmonary arterial endothelial cells (EC) exposed to cyclic strain. EC were subjected to 10% average strain at 60 cycles/min. Cyclic strain induced activation of extracellular signal-regulated kinase (ERK; 1.5-fold), c-Jun NH(2)-terminal protein kinase (JNK; 1.9-fold), and p38 (1. 5-fold) with a peak at 30 min. To investigate the functional role of the activated MAPKs, we analyzed cells after treatment with PD-98059, a specific ERK kinase inhibitor, or SB-203580, a catalytic inhibitor for p38, and after transient transfection with JNK(K-R), and MEKK(K-M) the respective catalytically inactive mutants of JNK1 and MAPK kinase kinase-1. Cyclic strain increased activator protein-1 (AP-1) binding activity, which was blocked by PD-98059 and SB-203580. Activity of AP-1-dependent luciferase reporter driven by 12-O-tetradecanoyl-phorbol-13-acetate-responsive element (TRE) was induced by cyclic strain, and this was attenuated by PD-98059, MEKK(K-M), JNK(K-R), and SB-203580. PD-98059 and SB-203850 did not inhibit cell alignment and migration induced by cyclic strain. MEKK(K-M) and JNK(K-R) transfection did not block cyclic strain-induced cell alignment. In conclusion, cyclic strain activates ERK, JNK, and p38, and their activation plays a role in transcriptional activation of AP-1/TRE but not in cell alignment and migration changes in bovine pulmonary arterial EC.

Outcomes of venous ulcer care: results of a longitudinal study.

A longitudinal study involving 81 patients with venous ulcers was conducted to explore the outcomes and cost of wound care in a home healthcare (HHC) setting and an outpatient care setting. Ulcers were managed with a saline gauze or hydrocolloid dressing and compression hosiery, or covered with an Unna's boot. Outcomes did not vary between physician's office and home care. Patients preferred home care, but costs and charges were much higher for HHC than for patients managed in the physician's office. Recurrence rates and costs varied greatly. Eighty-eight percent of ulcers in the saline dressing group did not heal or recurred compared to 21% of ulcers in the Unna's boot and 13% of ulcers in the hydrocolloid dressing group. The data also suggest hydrocolloid dressings are more cost-effective than Unna's boot or saline-gauze dressings. Controlled clinical studies to ascertain the cost-effectiveness of venous ulcer care in different patient care settings and the use of different treatment modalities, as well as care system oriented toward outcome for the patient rather than service, design, and distribution, are needed.

Nonrecurrent laryngeal nerve during carotid artery surgery: case report and literature review.

The anomalous position of a nonrecurrent inferior laryngeal nerve predisposes it to injury during surgery in the neck. We present the case of a patient who underwent a carotid endarterectomy in which a rare left nonrecurrent laryngeal nerve was found intraoperatively. This abnormality, which occurs much less often on the left than the right side of the neck, should be familiar to vascular surgeons. Historical, embryologic, and surgical significance of this anomaly is addressed.

Role of caveolin in hemodynamic force-mediated endothelial changes.

BACKGROUND: Caveolin has been shown to play an important role in signal transduction and nitric oxide synthase production. The purpose of this study was to investigate whether caveolin was tyrosine phosphorylated or activated by shear stress or cyclic strain in bovine aortic endothelial cells (BAECs). MATERIALS AND METHODS: BAECs were subjected to an average of 10% strain at a rate of 60 cycles/min or a laminar shear stress of 10 dyn/cm(2) for up to 4 h. Immunoblotting with anticaveolin antibody was performed to assess activation of caveolin. Coimmunoprecipitation of anticaveolin antibody with anti-tyrosine phosphorylation antibody was performed to detect the tyrosine phosphorylation of caveolin. RESULTS: Neither cyclic strain nor shear stress at physiologic levels altered the level of caveolin protein. Tyrosine phosphorylation of caveolin could not be observed at any time under either cyclic strain or shear stress condition. CONCLUSION: Although hemodynamic forces alter nitric oxide synthase production and activate signal transduction, caveolin levels or activity is not altered in endothelial cells exposed to shear stress or cyclic strain.

Inhibition of phosphatidylinositol 3-kinase and protein kinase C attenuates extracellular matrix protein-induced vascular smooth muscle cell chemotaxis.

PURPOSE: Intimal hyperplasia (IH), a significant cause of vascular reconstructive failure, is characterized by abnormal vascular smooth muscle cell (VSMC) migration, proliferation, and extracellular matrix (ECM) deposition. The ECM proteins, thrombospondin-1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) can induce VSMC migration; however, the cellular signaling pathways involved are not identical for each ECM protein. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are two enzymes that have been associated with VSMC migration. We sought to elucidate the roles of these enzymes in TSP-1-, Fn-, and Vn-stimulated VSMC migration. METHODS: Chemotaxis assays were performed by using a modified Boyden Chamber. TSP-1, Fn, or Vn (20 microg/mL) or serum-free media (SFM) was placed in the bottom wells of the chamber. Quiescent bovine aortic VSMC were preincubated with LY 294002 (100 micromol/L), a PI3K inhibitor, bisindolylmaleimide I (GF 109203X, 1 micromol/L), a PKC inhibitor, or in SFM alone for 30 minutes. VSMCs (50,000 cells per well) were then placed in the top wells of the chamber, and the assay was conducted for 4 hours at 37 degrees C. Results were recorded as the number of cells migrated per five fields (400x) and analyzed by means of the paired t test, with P value less than.05 considered to be significant (n = 3). RESULTS: The VSMC migration was significantly increased by TSP-1, Fn, and Vn. LY 294002 inhibited TSP-1-, Fn-, and Vn-stimulated VSMC migration (85% to 89%, P <.05). GF 109203X inhibited only TSP-1-stimulated migration (65%, P <.05). CONCLUSION: These results suggest that TSP-1-, Fn-, and Vn-stimulated migration is at least partially dependent on PI3K. However, only TSP-1 stimulated migration is at least partially dependent on PKC.

Gender does not impact infrainguinal vein bypass graft outcome.

BACKGROUND: The percentage of women requiring infrainguinal bypass graft operations continues to increase, whereas the effect of gender on postoperative outcome remains unclear. The purpose of this study was to assess the influence of gender on patient selection and outcome in patients requiring infrainguinal vein bypass grafting procedures. METHODS: This retrospective study reviewed 217 infrainguinal vein bypass grafts performed over an 8-year period. Medical records and patient interviews were used to determine study measures and outcomes. Gender and multiple covariables affecting patient survival were analyzed; postoperative complications and graft patencies were examined. Bivariate and life-table analyses were conducted, followed by multivariate analysis with the Cox proportional hazards model. RESULTS: No statistical differences existed between men and women for age, diabetes, cardiac disease, tobacco use, hypertension, stroke, renal disease, or prior contralateral bypass or major amputation. Women were more likely to be black (P =.014) and have a spliced vein graft (P =.035). No differences were noted between the 2 groups for 30-day morbidity rates-except women had more incisional complications (P =.01)-or for survival (P =.45), primary-patency (P =.57), secondary-patency (P =. 79), or limb-salvage rates (P =.40). Multivariate analysis showed that gender had no role in affecting survival rates. CONCLUSIONS: Gender does not affect graft patency, limb salvage, or survival rates. There should be no introduction of a gender bias into management of infrainguinal occlusive disease.

Mitogen-activated protein phosphorylation in endothelial cells exposed to hyperosmolar conditions.

The effect of hyperosmolarity on the induction of the mitogen-activated protein kinases (MAPK) was studied in bovine aortic endothelial cell (EC). Different types of agents were used to differentiate the effects of osmolarity from other variables. Hypertonic treatment with physiologically relevant levels of NaCl (350 mOsm/kg H(2)O) significantly increased the level of expression of p38 within 2 min, and ERK-1/2 and JNK after 10 min. The inductions peaked between 30 and 60 min and returned to baseline levels within 2 h. A similar pattern of induction occurred with ionic contrast agent. p38 induction by glucose and mannitol showed a similar pattern, although the level of ERK-1/2 phosphorylation was not as robust, and JNK was not induced by glucose. Urea did not affect the level of induction of the MAPK isoforms. It is concluded that MAPK plays an important role in hyperosmolality-induced signal transduction. Different osmotic agents induce MAPK expression differently. No MAPK induction with urea implies that cell shrinkage may be an important component of hyperosmolality-induced MAPK phosphorylation.

Localization of atherosclerosis: role of hemodynamics.

Atherosclerosis is a chronic disease attributed to risk factors that are systemic in nature. Yet the lesions involved do not occur in random fashion. The coronary arteries, the major branches of the aortic arch, and the abdominal aorta and its visceral and major lower extremity branches are particularly susceptible sites. Hemodynamic forces interacting with an active vascular endothelium are responsible for localizing lesions in a nonrandom pattern of distribution. Shear stress and cyclic circumferential strain are the predominant forces that have been characterized. The modification of endothelial cell structure and function by these mechanical forces sheds insight into the vasculature's propensity for atherogenesis.

Thrombospondin-1 regulation of smooth muscle cell chemotaxis is extracellular signal-regulated protein kinases 1/2 dependent.

BACKGROUND: Thrombospondin-1 (TSP-1), an extracellular matrix protein, induces vascular smooth muscle cell (VSMC) chemotaxis. We hypothesized that extracellular signal-regulated protein kinases 1/2 (ERK1/2), a pathway of the mitogen activated protein kinase (MAPK) family, is important in TSP-1-induced VSMC chemotaxis. METHODS: A modified Boyden chamber was used to assess chemotaxis. First, a concentration curve was performed to determine the level for optimal TSP-1-induced chemotaxis. Then quiescent VSMCs were preincubated (30 minutes) in serum-free medium, dimethyl sulfoxide (the inhibitor vehicle), or PD98059 (10 mumol/L, an upstream inhibitor of ERK1/2). VSMCs (50,000 cells/well) with the appropriate preincubation were placed in the top chamber. The bottom chamber contained TSP-1 (20 micrograms/mL) or serum-free medium. Results were recorded as cells/5 fields (400x). Then quiescent VSMCs were exposed to TSP-1 (20 micrograms/mL) for 0, 1, 5, 10, 30, 120, or 300 minutes. Platelet-derived growth factor (10 ng/mL) was the positive control for ERK1/2 activation. Western blot analysis was performed for activated ERK1/2. All comparisons were made by a paired t test (n = 3). RESULTS: TSP-1-induced chemotaxis peaks by a concentration of 20 micrograms/mL. PD98059 inhibited TSP-1-induced chemotaxis (P < .05). ERK1/2 was activated by TSP-1-stimulated VSMCs. CONCLUSIONS: TSP-1-stimulated VSMCs activated ERK1/2. An ERK1/2 inhibitor abolished chemotaxis, suggesting the functional importance of MAPK in TSP-1-induced VSMC chemotaxis.

Thrombospondin-1 induces activation of focal adhesion kinase in vascular smooth muscle cells.

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation and migration are important events in the development of intimal hyperplasia. Thrombospondin-1 (TSP-1), an extracellular matrix protein present in intimal hyperplastic lesions, has been shown to stimulate VSMC proliferation and migration. We hypothesized that the focal adhesion plaque, specifically the focal adhesion kinase (FAK) protein, may be important in the signal transduction pathway for TSP-1-induced VSMC migration. METHODS: Growth-arrested bovine aortic VSMCs were treated with TSP-1 (20 microg/mL) for set intervals (15, 30, and 120 minutes) and compared with VSMCs grown in serum-free medium (negative control) or in the presence of a known mitogen and chemotactic factor, platelet-derived growth factor (10 ng/mL; positive control). Crude cell lysates and anti-FAK immunoprecipitates were assayed for phosphotyrosine activity by means of antiphosphotyrosine immunoblotting. The blots were quantified by means of densitometric analysis. RESULTS: TSP-1 increased tyrosine phosphorylation of three protein bands of molecular weights 68, 125 (consistent with FAK), and 180 kDa. Immunoprecipitation with FAK antibody, followed by antiphosphotyrosine immunoblotting, indicated that there was an increase in tyrosine phosphorylation of FAK at 15, 30, and 120 minutes in the TSP-1-treated groups (P <.05). CONCLUSION: Tyrosine phosphorylation of FAK is induced by TSP-1 stimulated VSMCs. This suggests an outside-inside signaling pathway by which TSP-1 stimulates VSMC migration.

Deep venous thrombosis and pulmonary embolism in bilateral lower-extremity amputee patients.

OBJECTIVE: To determine the incidence of deep venous thrombosis and pulmonary emboli and the value of an inferior vena cava filter in patients with bilateral lower-extremity amputations, and to determine the incidence of pulmonary emboli after filter placement. DESIGN: Retrospective study with a follow-up of 3 to 64 months. SETTING: Inner-city university hospital. PATIENTS: Twenty-seven consecutive patients with bilateral lower-limb amputation. RESULTS: Age, sex, and race were assessed, and had no impact on the incidence of pulmonary embolus in these patients with lower-extremity amputation. CONCLUSION: No clinical objective evidence of pulmonary emboli occurred after placement of an inferior vena caval filter.

Use of venous duplex scans to evaluate symptoms of deep vein thrombosis: an analysis of ultrasound usage by various medical specialties.

In today's environment of cost-containment and utilization management, duplex ultrasound is often overused to evaluate symptoms of deep venous thrombosis/thrombophlebitis (DVT), reflecting the low diagnostic yield of such studies. We investigated the use of venous duplex scans by various medical specialties to determine whether a tendency exists to overuse this diagnostic tool by one specialty compared with others and to assess the cost-effectiveness of ordering this test for acute venous disease changes. We retrospectively reviewed the results of venous duplex ultrasound studies for 330 consecutive patients with suspected DVT for a 1-year period. Our analysis showed that 51 of 330 (16%) of all duplex scans ordered in our institution were positive for DVT. Internists, as a group, ordered 185 studies of which 26 were positive (14%). The surgeons' requests for duplex studies resulted in 23 of 137 (17%) confirmed positive studies. The overall positive examination rate was 16 per cent, which is not only suboptimal as a diagnostic tool, but also cumbersome with regard to health care cost-containment.

What is the paradigm: hospital or home health care for pressure ulcers?

A home health care (HHC) referral should link the patient in a cost-effective fashion to the physician, home care, and instructions regarding ulcer management. Twenty-one patients (mean age, 74.6 years) had stage III pressure ulcers (<100 cm2) and an involved family member at home. Risk and contributing factors included cardiac disease (n = 9), hypertension (n = 14), end-stage renal disease (n = 7), smoking (n = 11), diabetes (n = 8), chronic brain syndrome (n = 14), cerebrovascular accident (n = 5), and above-the-knee amputation (n = 2). Treatment regimens included standard wound care, pressure relief and, where appropriate, culture-specific antibiotics, as well as a rehabilitation program. Home care progressively decreased the frequency of the nurse HHC and physician office visits. Resolution of the pressure ulcer varied from 6 to 32 weeks. Only two patients had progression of their wound and required hospital readmission. The billable fees included: 1) an office visit, $30.00 (medicare reimbursement, $14.00); 2) the HHC nurse visit, $159.00 (medicare reimbursement, $105.00); 3) supplies, $75.00 to $150.00/week (variable reimbursement); 4) hospitalization, $400.00 to $900.00/day; and 5) a chronic-care bed, $400.00 to $750.00/day. HHC, given a responsible support team and an involved family member, was more socially and financially acceptable than an inpatient facility. Intermittent physician visits with HHC proved safe and reliable, with 90 per cent successfully healing their wounds.