Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction.

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.

In-stent recurrent stenosis after carotid artery stenting: life table analysis and clinical relevance.

OBJECTIVES: Carotid artery stenting has been proposed as an alternative to carotid endarterectomy in cerebral revascularization. Although early results from several centers have been encouraging, concerns remain regarding long-term durability of carotid artery stenting. We report the incidence, characteristics, and management of in-stent recurrent stenosis after long-term follow-up of carotid artery stenting. METHODS: Carotid artery stenting (n = 122) was performed in 118 patients between September 1996 and March 2003. Indications included recurrent stenosis after previous carotid endarterectomy (66%), primary lesions in patients at high-risk (29%), and previous ipsilateral cervical radiation therapy (5%). Fifty-five percent of patients had asymptomatic stenosis; 45% had symptomatic lesions. Each patient was followed up with serial duplex ultrasound scanning. Selective angiography and repeat intervention were performed when duplex ultrasound scans demonstrated 80% or greater in-stent recurrent stenosis. Data were prospectively recorded, and were statistically analyzed with the Kaplan-Meier method and log-rank test. RESULTS: Carotid artery stenting was performed successfully in all cases, with the WallStent or Acculink carotid stent. Thirty-day stroke and death rate was 3.3%, attributable to retinal infarction (n = 1), hemispheric stroke (n = 1), and death (n = 2). Over follow-up of 1 to 74 months (mean, 18.8 months), 22 patients had in-stent recurrent stenosis (40%-59%, n = 11; 60%-79%, n = 6; > or =80%, n = 5), which occurred within 18 months of carotid artery stenting in 13 patients (60%). None of the patients with in-stent recurrent stenosis exhibited neurologic symptoms. Life table analysis and Kaplan-Meier curves predicted cumulative in-stent recurrent stenosis 80% or greater in 6.4% of patients at 60 months. Three of five in-stent recurrent stenoses occurred within 15 months of carotid artery stenting, and one each occurred at 20 and 47 months, respectively. Repeat angioplasty was performed once in 3 patients and three times in 1 patient, and repeat stenting in 1 patient, without complications. One of these patients demonstrated asymptomatic internal carotid artery occlusion 1 year after repeat intervention. CONCLUSIONS: Carotid artery stenting can be performed with a low incidence of periprocedural complications. The cumulative incidence of clinically significant in-stent recurrent stenosis (> or =80%) over 5 years is low (6.4%). In-stent restenosis was not associated with neurologic symptoms in the 5 patients noted in this cohort. Most instances of in-stent recurrent stenosis occur early after carotid artery stenting, and can be managed successfully with endovascular techniques.

Does severe venous insufficiency have a different etiology in the morbidly obese? Is it venous?

OBJECTIVE: Chronic venous insufficiency (CVI) is the most common cause of leg ulcers. Patients with morbid obesity are remarkable for particularly recalcitrant ulcers. Because obesity is not specifically incorporated in CEAP or other venous scoring systems, we sought to characterize this group of patients more completely. METHODS: Patients with severe CVI (CEAP clinical class, 4, 5, and 6), and class III obesity (body mass index [BMI], >40) were reviewed. Findings from clinical and duplex ultrasound scan (DU) examinations were compared with the CEAP classification, its adjunctive venous clinical severity score, and sensory thresholds. RESULTS: A review of clinic records identified 20 ambulatory patients with a mean age of 62 years, a mean BMI of 52, and a mean weight of 164 kg (361 lbs); all but one had bilateral symptoms. No evidence of venous insufficiency was detected with DU in 24 of the 39 limbs. Although some valvular incompetence was detected with DU in 15 of 39 limbs, these abnormalities were widely dispersed between 28 sites; eight limbs had findings at only one site. Ulceration (mean area, 29 cm(2)) was present in 25 limbs and necessitated 7 months for healing; 13 (52%) recurred at least once during a mean observation period of 36 months. The mean sensory threshold of 5.21 exceeded current risk thresholds used in diabetic screening programs. The distribution of CEAP clinical class was C4 (n = 14), C5 (n = 14), and C6 (n = 11). Increasing CEAP class correlated with an increased mean BMI of 47, 52, and 56, respectively (P <.01). CEAP also correlated with a rising mean venous clinical severity score of 10, 11, and 15, respectively (P <.05). CONCLUSION: Patients with class III obesity had severe limb symptoms, typical of CVI, but approximately two thirds of the limbs had no anatomic evidence of venous disease. The association of increasing limb symptoms with increasing obesity suggested that the obesity itself contributes to the morbidity.

p42/44MAPK regulates baseline permeability and cGMP-induced hyperpermeability in endothelial cells.

We tested the hypothesis that p42/44MAPK and p38MAPK (mitogen-activated protein kinases; MAPK) signaling pathways regulate endothelial cell permeability to macromolecules. Passage 2-4 human umbilical vein endothelial cells (HUVEC) were grown to confluence on fibronectin-coated Snapwell membranes. The flux of fluorescein isothiocyanate-labeled dextran-70 across the HUVEC monolayers served to determine permeability. Application of 1 mM 8-bromo 3' 5'-cyclic guanosine monophosphate (8-Br-cGMP) increased permeability from 7.0 +/- 1.6 x 10(-6) to 12.5 +/- 2.8 x 10(-6) cm/s (P < 0.05). Pretreatment of HUVEC for 60 min with a selective p42/44MAPK inhibitor (AG126 at 2.7 and 27 microM) blocked 8-Br-cGMP-induced hyperpermeability. However, inhibition of p38MAPK (SB203580 at 0.6 microM) did not influence the cGMP-induced hyperpermeability response. AG126, administered at 27 microM, decreased baseline permeability from 7.9 +/- 0.5 x 10(-6) to 5.9 +/- 0.5 x 10(-6) cm/s (P < 0.05). Our results indicate that the p42/44MAPK signaling pathway is important in the regulation of baseline permeability and cGMP-induced hyperpermeability.