Centre for Molecular Biosciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry, UK.
J Physiol. 2011 Oct 1;589(Pt 19):4681-96. doi: 10.1113/jphysiol.2011.208355. Epub 2011 Jul 18.
Regeneration of injured tissue is a dynamic process, critically dependent on the formation of new blood vessels and restructuring of the nascent plexus. Endothelial barrier function, a functional correlate of vascular restructuring and maturation, was quantified via intravital microscopic analysis of 150 kDa FITC-dextran-perfused blood vessels within discrete wounds created in the panniculus carnosus (PC) muscle of dorsal skinfold chamber (DSC) preparations in mice. Time to recovery of half-peak fluorescence intensity (t(1/2)) within individual vessel segments in three functional regions of the wound (pre-existing vessels, angiogenic plexus and blind-ended vessels (BEVs)) was quantified using in vivo fluorescence recovery after photobleaching (FRAP) and linear regression analysis of recovery profiles. Plasma flux across the walls of new vessel segments, particularly BEVs, was greater than that of pre-existing vessels at days 5-7 after injury (P < 0.05). TNP-470 reduced the permeability of BEVs at the leading edge of the advancing vascular plexus as measured by the decrease in luminal t(1/2) (P < 0.05), confirming the utility of FRAP as a quantitative measure of endothelial barrier function. Furthermore, these data are suggestive of a role for TNP-470 in selection for less leaky vascular segments within healing wounds. Increased FITC-dextran leakage was observed from pre-existing vessels after treatment with TNP-470 (P < 0.05), consistent with induction of transient vascular damage, although the significance of this finding is unclear. Using in vivo FRAP this study demonstrates the relationship between temporal changes in microvascular macromolecular flux and the morphology of maturing vascular segments. This combination of techniques may be useful to assess the therapeutic potential of angiogenic agents in restoring pre-injury levels of endothelial barrier function, following the establishment of a functional vascular plexus such as in models of wounding or tumour development.
组织损伤的再生是一个动态过程，严重依赖于新血管的形成和新生丛的重构。血管重构和成熟的功能相关物是内皮屏障功能，通过在小鼠背部皮肤囊（DSC）制备的背阔肌（PC）肌肉中离散伤口内用 150 kDa FITC-葡聚糖灌注的血管进行活体显微镜分析来量化。使用活体荧光恢复后漂白（FRAP）和恢复曲线的线性回归分析来量化个体血管段中半峰荧光强度（t(1/2)）恢复的时间，在伤口的三个功能区域（预先存在的血管、血管生成丛和盲端血管（BEVs））内的单个血管段。在损伤后 5-7 天，新血管段，特别是 BEVs，的血浆通量超过预先存在的血管（P < 0.05）。TNP-470 通过减少管腔 t(1/2)来减少血管生成丛中前沿的 BEVs 的通透性（P < 0.05），证实了 FRAP 作为内皮屏障功能定量测量的有用性。此外，这些数据表明 TNP-470 在选择愈合伤口中渗漏性较低的血管段方面具有作用。在用 TNP-470 处理后，预先存在的血管中观察到 FITC-葡聚糖渗漏增加（P < 0.05），这与诱导短暂性血管损伤一致，尽管这一发现的意义尚不清楚。本研究通过活体 FRAP 证明了微血管大分子通量的时间变化与成熟血管段形态之间的关系。这种技术组合可能有助于评估血管生成剂在建立功能性血管丛（如在创伤或肿瘤发展模型中）后恢复内皮屏障功能的治疗潜力。
