VEGF-mediated elevated intracellular calcium and angiogenesis in human microvascular endothelial cells in vitro are inhibited by dominant negative TRPC6.

OBJECTIVE: Vascular endothelial growth factor (VEGF)-induced vascular permeability has been shown to be dependent on calcium influx, possibly through a transient receptor potential cation channel (TRPC)-mediated cation channel with properties of the TRPC3/6/7 subfamily. To investigate further the involvement of this subfamily, we determined the effects of dominant negative TRPC6 overexpression on VEGF-mediated changes of human microvascular endothelial cell (HMVEC) calcium, proliferation, migration, and sprouting. METHODS: Cytoplasmic calcium concentration was estimated by fura-2 fluorescence spectrophotometry, migration by Boyden chamber assay, sprouting by immunofluorescence imaging of stimulated endothelial cells, and proliferation by flow cytometry. RESULTS: Overexpression of a dominant negative TRPC6 construct in HMVECs inhibited the VEGF-mediated increases in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild-type TRPC6 construct increased the proliferation and migration of HMVECs. CONCLUSIONS: TRPC6 is an obligatory component of cation channels required for the VEGF-mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis.

Successful perioperative management of factor X deficiency associated with primary amyloidosis.

Acquired bleeding abnormalities are common in patients with primary amyloid light-chain amyloidosis. Factor X deficiency is the most common coagulopathy associated with life-threatening hemorrhagic complications when surgery is indicated. Fresh-frozen plasma (FFP) or prothrombin complex concentrates (PCCs) are the most frequently used blood products in this disease; however, FFP is often ineffective in controlling bleeding and PCCs have a significant risk of thrombosis when used intraoperatively. This report describes a patient with primary amyloidosis and factor X deficiency who underwent hemicolectomy with preoperative and intraoperative administration of recombinant human factor VIIa and postoperative administration of Bebulin (a PCC that contains the highest concentration of factor X). The management was successful with no signs of bleeding postoperatively. To our knowledge, few reports of successful perioperative management of factor X deficiency have been published to date. This is the first case report using recombinant human factor VIIa and Bebulin in the perioperative management of factor X deficiency associated with primary amyloidosis. Recombinant human factor VIIa and Bebulin may allow for successful perioperative management of bleeding disorders in patients with primary amyloidosis.

Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx.

AIMS: In this study, we wished to determine whether angiopoietin-1 (Ang1) modified the permeability coefficients of non-inflamed, intact continuous, and fenestrated microvessels in vivo and to elucidate the underlying cellular mechanisms. METHODS AND RESULTS: Permeability coefficients were measured using the Landis-Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (L(P): hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (sigma: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both L(P) and sigma. Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in L(P) (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin. CONCLUSION: Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects.

Transient osmotic absorption of fluid in microvessels exposed to low concentrations of dimethyl sulfoxide.

Dimethyl Sulfoxide (DMSO) is a common solvent for pharmacological agents. It is a small, lipophilic molecule thought to be relatively highly permeable through the cell membrane. While measuring the effect of low concentrations of DMSO (0.05-0.5% v/v) on capillary hydraulic conductivity as a vehicle control for pharmacological agents, the authors noticed what appeared to be an unusual transient absorption of fluid across the vessel wall. This absorption occurred during occlusion of the vessel, but dissipated quickly (1.7-8.6 s). The transient reabsorption reappeared upon each successive occlusion. To determine the nature of this transient absorption, the authors have measured the effect of increasing the pressure of the perfusing solution, of the concentration and time of perfusion of DMSO, and of superfusing the DMSO. They found that the absorption rate, but not the filtration rate, was concentration dependent, and was significantly correlated with the osmotic pressure of the DMSO. Moreover, the time taken for completion of the transient, i.e., time to reversal of flow, was inversely proportional to the hydraulic conductivity of the vessel. Furthermore, the transient absorption could be reduced and eventually abolished by increasing the hydrostatic pressure. These results strongly suggested that perfusion with low concentrations of DMSO could set up a significant osmotic pressure gradient across the vessel wall. This proposed mechanism for the absorption was confirmed by the measurement of a significant osmotic reflection coefficient of the vessel wall to DMSO (0.11 +/- 0.01). Relatively low concentrations (0.05-0.5%) of DMSO were therefore able to stimulate a significant osmotic transient across the blood vessel walls.