We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.

IntroductionDapagliflozin is a selective sodium glucose co-transporter 2 inhibitor that improves glycemic control and reduces body weight and systolic blood pressure in patients with type 2 diabetes mellitus (T2DM). Dapagliflozin is effective and well tolerated over 12–24 weeks in Japanese patients with T2DM. In this study, the safety and efficacy of dapagliflozin administered as monotherapy and combination therapy were assessed over 52 weeks in Japanese patients with T2DM.MethodsThis was a 52-week open-label Phase 3 study consisting of a single treatment arm with no comparator. Dapagliflozin was administered as monotherapy (n = 249) or combination therapy (n = 479) with existing antihyperglycemic agents (sulfonylurea, glinides, metformin, alpha-glucosidase inhibitors, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, or glucagon-like peptide-1 receptor agonists) to Japanese patients with T2DM and inadequate glycemic control for 52 weeks. Treatment with dapagliflozin was initiated at 5 mg/day and titrated to 10 mg/day as required.ResultsDapagliflozin administered as monotherapy or combination therapy was well tolerated. The frequency of adverse events (AEs) over 52 weeks was similar between monotherapy (79.1%) and combination therapy (72.4%) groups, and AEs were mostly mild or moderate. The incidence of hypoglycemia at 52 weeks was 2.4% in the monotherapy group and 4.0% in the combination therapy group. In patients receiving dapagliflozin as monotherapy or combination therapy, reductions from baseline to week 52 were observed in glycosylated hemoglobin (HbA1c) (−0.7% in both groups), weight (−2.6 and −2.1 kg, respectively), and systolic blood pressure (−5.2 mmHg and −3.9 mmHg). In patients with insufficient response to 5 mg/day, dapagliflozin was increased to 10 mg/day, and a further decrease in HbA1c from the pre-titration value was observed in both groups.ConclusionDapagliflozin was well tolerated and effective as monotherapy or combination therapy in Japanese patients with T2DM over 52 weeks.Electronic supplementary materialThe online version of this article (doi:10.1007/s13300-014-0086-7) contains supplementary material, which is available to authorized users.

Endothelial cells (ECs) are exposed to repetitive cyclic strain (CS) in vivo by the beating heart. The aim of this study was to assess the influence of CS amplitude and/or frequency on EC proliferation and survival and to determine the role of AKT in CS-induced EC proliferation and survival. Cultured bovine aortic ECs were exposed to 10% strain at a frequency of 60 (60 cpm-10%) or 100 (100 cpm-10%) cycles/min or 15.6% strain at a frequency of 60 cycles/min (60 cpm-15.6%). AKT, glycogen synthase kinase (GSK)-3beta, BAD, and cleaved caspase-3 were activated by CS in ECs. Increasing the magnitude or frequency of strain resulted in an earlier phosphorylation of GSK-3beta, although the magnitude of phosphorylation was similar. After CS at 60 cpm-10% for 24 h, the number of nontransfected ECs was significantly increased by 8.5% (P < 0.05). We found that the number of apoptotic ECs was slightly decreased with exposure to CS. ECs transfected with kinase-dead AKT (KA179) as well as plasmids containing a point mutation in the pleckstrin homology domain of AKT (RC25) not only prevented AKT, GSK-3beta, and BAD phosphorylation but also inhibited the CS-induced increase in cell number as well as the CS-induced protection against apoptosis (both P < 0.05). The ratio of 5'-bromo-2'-deoxyuridine-positive cells was increased when ECs transfected with RC25 and KA179 as well as nontransfected ECs and ECs transfected with Lipofectamine 2000 were exposed to CS. We conclude that AKT is important in enhancing the survival of ECs exposed to CS but is not involved in EC proliferation.

Fluid shear stress is thought to be important in maintaining the phenotype of endothelial cells (ECs) in vivo. The purpose of the study was to determine the effect of varying levels of laminar shear stress on EC elongation and alignment and the role of p38 mitogen-activated protein kinase (MAPK) on the morphologic change induced by shear stress. Cultured bovine aortic ECs were subjected to 1, 4, 7, 14, or 20 dyne/cm(2) laminar steady shear stress. On morphometric analysis of static ECs, the average orientation angle was 41 degrees , whereas after 24 h shear stress at 1, 4, 7, 14, and 20 dyne/cm(2) the angles were 34 degrees, 33 degrees, 16 degrees, 11 degrees, and 10 degrees, respectively. The shape index of static ECs was 0.76, whereas the indexes of ECs exposed to shear stress were 0.72, 0.72, 0.65, 0.50, and 0.47, respectively. The time and the magnitude of activation of p38 MAPK were dependent on the level of shear stress. The results indicate that a minimum shear stress of 7 to 14 dynes/cm(2) is necessary for cell alignment and elongation and this correlates with activity of p38 MAPK. ECs exposed to shear stress in the presence of the p38 MAPK inhibitor SB-203580 did not orient in any manner and the shape index was similar to the static cells.