Exposing a vein to altered hemodynamics by creating an arteriovenous (AV) shunt evokes considerable vessel formation that may be of therapeutic potential. However, it is unclear whether the introduction of oscillatory flow and/or flow increase is decisive. To distinguish between these mechanical stimuli we grafted a femoral vein into the arterial flow pathway of the contralateral limb in rats creating an arterioarterial (AA) loop (n = 7). Alternatively, we connected the femoral artery and vein using the vein graft, whereby we created an AV-loop (n = 27). Vessel loops were embedded in a fibrin filled chamber and blood flow was measured by means of flow probes immediately after surgery (day 0) and 15 days after loop creation. On day 15, animals were sacrificed and angiogenesis was evaluated using μCT and histological analysis. Mean flow increased from 0.5 to 2.4 mL/min and was elevated throughout the cardiac cycle at day 0 in AV-loops whereas, as expected, it remained unchanged in AA-loops. Flow in AV-loops decreased with time, and was at day 15 not different from untreated femoral vessels or AA-loop grafts. Pulsatile flow oscillations were similar in AV-and AA-loops at day 0. The flow amplitude amounted to ~1.3 mL/min which was comparable to values in untreated arteries. Flow amplitude remained constant in AA-loops, whereas it decreased in AV-loops (day 15: 0.4 mL/min). A large number of newly formed vessels were present in AV-loops at day 15 arising from the grafted vein. In marked contrast, angiogenesis originating from the grafted vein was absent in AA-loops. We conclude that exposure to substantially increased flow is required to initiate angiogenesis in grafted veins, whereas selective enhancement of pulsatile flow is unable to do so. This suggests that indeed flow and most likely wall shear stress is decisive to initiate formation of vessels in this hemodynamically driven angiogenesis model.

Gap junctions are involved in vascular growth and their expression pattern is modulated in response to hemodynamic conditions. They are clusters of intercellular channels formed by connexins (Cx) of which four subtypes are expressed in the cardiovascular system, namely Cx37, Cx40, Cx43 and Cx45. We hypothesize that high flow conditions affect vascular expression of Cx in vivo. To test this hypothesis, flow hemodynamics and subsequent changes in vascular expression of Cx were studied in an angioinductive rat arteriovenous (AV) loop model. Fifteen days after interposition of a femoral vein graft between femoral artery and vein encased in a fibrin-filled chamber strong neovascularization was evident that emerged predominantly from the graft. Blood flow through the grafted vessel was enhanced ∼4.5-fold accompanied by increased pulsatility exceeding arterial levels. Whereas Cx43 protein expression in the femoral vein is negligible at physiologic flow conditions as judged by immunostaining its expression was enhanced in the endothelium of the venous graft exposed to these hemodynamic changes for 5 days. This was most likely due to enhanced transcription since Cx43 mRNA increased likewise, whereas Cx37 mRNA expression remained unaffected and Cx40 mRNA was reduced. Although enhanced Cx43 expression in regions of high flow in vivo has already been demonstrated, the arteriovenous graft used in the present study provides a reliable model to verify an association between Cx43 expression and high flow conditions in vivo that was selective for this Cx. We conclude that enhancement of blood flow and its oscillation possibly associated with the transition from laminar to more turbulent flow induces Cx43 expression in a vein serving as an AV loop. It is tempting to speculate that this upregulation is involved in the vessel formation occuring in this model as Cx43 was suggested to be involved in angiogenesis.

BackgroundThe arteriovenous (AV) loop model enables axial vascularization to gain a functional microcirculatory system in tissue engineering constructs in vivo. These constructs might replace surgical flaps for the treatment of complex wounds in the future. Today, free flaps are often exposed to high-dose radiation after defect coverage, according to guideline-oriented treatment plans. Vascular response of AV loop-based constructs has not been evaluated after radiation, although it is of particular importance. It is further unclear whether the interposed venous AV loop graft is crucial for the induction of angiogenesis.Material/MethodsWe exposed the grafted vein to a single radiation dose of 2 Gy prior to loop construction to alter intrinsic and angio-inductive properties specifically within the graft. Vessel loops were embedded in a fibrin-filled chamber for 15 days and radiation-induced effects on flow-mediated vascularization were assessed by micro-CT and two-dimensional histological analysis.ResultsVessel amount was significantly impaired when an irradiated vein graft was used for AV loop construction. However, vessel growth and differentiation were still present. In contrast to vessel density, which was homogeneously diminished in constructs containing irradiated veins, vessel diameter was primarily decreased in the more peripheral regions.ConclusionsVascular luminal sprouts were significantly diminished in irradiated venous grafts, suggesting that the interposing vein constitutes a vital part of the AV loop model and is essential to initiate flow-mediate angiogenesis. These results add to the current understanding of AV loop-based neovascularization and suggest clinical implications for patients requiring combined AV loop-based tissue transfer and adjuvant radiotherapy.