eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering.

This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose-derived stem cells (ASC) differentiated into endothelial-like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO-producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non-thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial-like lineage imparts a non-thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications

Role of angiotensin II type 1A receptor phosphorylation, phospholipase D, and extracellular calcium in isoform-specific protein kinase C membrane translocation responses

The angiotensin II type 1A receptor (AT(1A)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1A)R is coupled via G(q) to the activation of phospholipase C, the hydrolysis of phosphoinositides, release of calcium from intracellular stores, and the activation of protein kinase C (PKC). We show here that PKC beta I and PKC beta II exhibit different membrane translocation patterns in response to AT(1A)R agonist activation. Whereas PKC beta II translocation to the membrane is transient, PKC beta I displays additional translocation responses: persistent membrane localization and oscillations between the membrane and cytosol following agonist removal. The initial translocation of PKC beta I requires the release of calcium from intracellular stores and the activation of phospholipase C, but persistent membrane localization is dependent upon extracellular calcium influx. The mutation of any of the three PKC phosphorylation consensus sites (Ser-331, Ser-338, and Ser-348) localized within the AT(1A)R C-tail significantly increases the probability that persistent increases in diacylglycerol levels and PKC beta I translocation responses will be observed. The persistent increase in AT(1A)R-mediated diacylglycerol formation is mediated by the activation of phospholipase D. Although the persistent PKC beta I membrane translocation response is absolutely dependent upon the PKC activity-dependent recruitment of an extracellular calcium current, it does not require the activation of phospholipase D. Taken together, we show that the patterning of AT(1A)R second messenger response patterns is regulated by heterologous desensitization and PKC isoform substrate specificity

Vascular control for a forequarter amputation of a massive fungating humeral osteosarcoma

Forequarter amputation is a radical operation performed for treatment of malignant neoplasms of the shoulder girdle not amenable to limb salvage. Traditional approaches involve bone and soft tissue resection, followed by ligation of the axillary vessels. We describe a technique to minimize blood loss whereby control of the subclavian vessels is performed before amputation of a large tumor associated with extensive venous congestion. A 34-year-old man presented with proximal humeral osteosarcoma. Surgery involved claviculectomy to facilitate vascular control of the subclavian vessels, followed by guillotine amputation at the proximal upper arm level and completion of the amputation as conventionally described