Vivian Gahtan

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.

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 H2O) 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. J. Cell. Biochem. 76:567–571, 2000.

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

BACKGROUNDnThrombospondin-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.nnnMETHODSnA 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).nnnRESULTSnTSP-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.nnnCONCLUSIONSnTSP-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 matrix metalloproteinase-2 activation in vascular smooth muscle cells

INTRODUCTIONnThrombospondin-1 (TSP-1), an extracellular matrix (ECM) glycoprotein, is associated with a variety of cellular processes relevant to atherosclerosis and intimal hyperplasia, including vascular smooth muscle cell (VSMC) migration. Matrix metalloproteinase-2 (MMP2) is associated with basement membrane and ECM degradation, important processes for cell migration. We hypothesized that TSP-1 modulates MMP2 activity in VSMCs and is critical for VSMC migration.nnnMETHODSnQuiescent bovine aortic VSMCs (48 hours) were incubated in serum-free media (SFM) with or without TSP-1 (10 or 20 microg/mL). Gelatinase activity was measured with zymography to determine pro-MMP2 and MMP2 activity. MMP2 messenger RNA expression was determined with Northern blot analysis. Invasion assays were performed. A binding assay was used to determine the specificity of TSP-1 binding to MMP2. Blots were quantified with densitometry, and all comparisons were made with a paired t test.nnnRESULTSnTSP-1 induced production of activated forms of MMP2, as well as upregulation of pro-MMP2. MMP2 mRNA was upregulated 1.7-fold by TSP-1 at 10 and 20 microg/mL. GM6001, an MMP inhibitor, inhibited VSMC migration across the matrix barrier, whereas migration that occurred in the absence of the matrix barrier was unaffected. With a binding assay, TSP-1 interacted physically with MMP2, and TSP-1-bound MMP2 showed the strongest binding activity in comparison with collagen I, fibronectin, and elastin.nnnCONCLUSIONnTSP-1 induced MMP2 activation through transcriptional and posttranslational mechanisms. These findings imply that MMP2 activation is relevant to the mechanism of TSP-1-induced VSMC migration.

Carotid endarterectomy outcome is not affected in patients with a contralateral carotid artery occlusion

BACKGROUNDnThe purpose of this retrospective patient/control patient study was to determine perioperative risk and long-term benefits of carotid endarterectomy contralateral to an occluded internal carotid artery.nnnMETHODSnThirty-seven patients undergoing carotid endarterectomy contralateral to an occluded internal carotid artery were each paired with two control group patients (n = 74) undergoing carotid endarterectomy contralateral to a patent internal carotid artery. Patients preferentially underwent electroencephalographic monitoring, selective shunting, and patch angioplasty for vessel closure.nnnRESULTSnThe perioperative rate of stroke or death was 5% (n = 2) in the occluded group and 3% (n = 2) in the control group. Ninety-two percent of the occluded group and 96% of the control patients were stroke-free over a mean follow-up of 23.8 and 27.2 months, respectively. No statistical difference was noted between groups for perioperative rate of stroke or death (P = 0.60), mean stroke-free rates (P = 0.37), stroke rate by life-table analysis (P = 0.33), or survival by life-table analysis (P = 0.43).nnnCONCLUSIONSnPatients who have carotid endarterectomy performed contralateral to an occluded internal carotid artery showed no difference for perioperative stroke or death, late stroke, or survival.

Thrombospondin‐1‐induced vascular smooth muscle cell chemotaxis: The role of the type 3 repeat and carboxyl terminal domains

Thrombospondin‐1 (TSP‐1), an acute phase reactant implicated in vascular disease, is a matricellular glycoprotein with six domains that confer different functions. The authors have shown TSP‐1 induces vascular smooth muscle cell (VSMC) chemotaxis via extracellular signal‐regulated kinases‐1 and ‐2 (ERK) and p38 kinase (p38) and that a fusion protein of the carboxyl terminal (COOH) and type 3 repeat (T3) domains independently induce VSMC chemotaxis. The purpose of this study was to determine whether COOH‐, T3‐induced VSMC chemotaxis, or both, is dependent upon ERK or p38 activation. To determine if the T3, COOH, or type 2 repeat domain (T2, control domain not associated with chemotaxis) activate ERK, p38, or both, VSMCs were exposed to each fusion protein (20 μg/ml for 15, 30, 60, or 120 min), serum‐free media (SFM, negative control), or TSP‐1 (20 μg/ml for 30 min, positive control). Western immunoblotting was performed for activation studies. Using a microchemotaxis chamber, VSMCs pre‐incubated in SFM, DMSO (vehicle control), PD98059 (10 μM), or SB202190 (10 μM) were exposed to each domain, TSP‐1, or SFM. After 4 h (37°C), migrated VSMCs were recorded as cells/five fields (400u2009×) and analyzed by paired t‐test. ERK was activated by T2, T3, and COOH. However, p38 was activated by T3 and COOH, but not T2. T3 and COOH‐induced VSMC chemotaxis were inhibited by PD98059 or SB202190, but more completely by SB202190. The T2 domain had no effect on VSMC chemotaxis. These results suggest activation of the p38 pathway may be more specific than ERK for COOH‐ and T3‐induced VSMC chemotaxis.

Predictors for the Healing of Transmetatarsal Amputations: Retrospective Study of 91 Amputations

This retrospective study reviewed 80 consecutive patients (mean age 62 years; range 21–91 years) who underwent 91 transmetatarsal amputations (TMAs) between 1995 and 2003. The mean follow-up was 12 ± 1.36 months. Sixty-two TMAs healed initially (group 1), whereas 29 TMAs did not heal by 3 months (group 2). At the final examination, in groups 1 and 2, 63 of 91 (69%) limbs were healed. Of the 28 limbs that did not heal, 25 of 28 (89%) required further proximal amputation. Initial healing correlated significantly with the ability to ambulate (p < .0001) and overall limb salvage (p < .0001). In group 1, 20 of 27 (74%) limbs that were revascularized healed (p = .0336). Nonhealing amputations were associated with end-stage renal disease (13 of 19; 68%) (p = .0209) and leukocytosis (13 of 19; 68%) (p = .0052).

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

OBJECTIVEnTo 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.nnnDESIGNnRetrospective study with a follow-up of 3 to 64 months.nnnSETTINGnInner-city university hospital.nnnPATIENTSnTwenty-seven consecutive patients with bilateral lower-limb amputation.nnnRESULTSnAge, sex, and race were assessed, and had no impact on the incidence of pulmonary embolus in these patients with lower-extremity amputation.nnnCONCLUSIONnNo clinical objective evidence of pulmonary emboli occurred after placement of an inferior vena caval filter.