Endothelial Maturity as a Function of Shear Stress: A Novel, Non‐invasive Intravital Imaging Approach

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Permanent neovessel stabilization after angiogenesis is essential for functional tissue recovery during wound healing. The transition from an angiogenic sprout to a functional capillary segment requires vessel lumen formation, pericyte (PC) recruitment, and mature differentiation of the endothelium. Expression of Tie2 by endothelial cells (ECs) has been purported to be a hallmark of maturation and vessel integrity. Prior in vitro studies have suggested that shear stress following the onset of blood flow after lumen formation might stimulate EC maturity. However, no study has been able to track EC identity and blood flow in an in vivo, adult model of angiogenesis.Elucidating the dynamics of angiogenesis requires the ability to make serial, in vivo observations of multicellular processes and vessel functionality. The first objective of this study was to create an intravital imaging protocol to serially (1) observe capillary EC‐PC interactions, (2) quantify transgenic gene‐driven GFP‐expression of EC markers, and (3) quantify blood flow (to calculate shear stress). Using our novel toolset, we investigated our second objective: to determine if blood flow precedes EC maturation and if a change in blood flow‐induced shear stress is sufficient to cause a change in EC Tie2 expression.In order to induce neovascularization, we used the cornea alkali burn model. This stimulated angiogenesis into the avascular cornea from the surrounding limbal vessels. We used endogenously labeled Tie2‐GFP × NG2‐DsRed dual fluorescent‐reporter mice to visualize ECs and PCs, respectively. Using intravital confocal microscopy, we observed angiogenic and stable neovessels at single‐cell resolution. We coupled this technique with photoacoustic microscopy (PAM), an imaging method that uses optical and acoustical approaches to obtain high‐resolution images of blood flow and oxygen saturation in microvascular networks. Finally, by selectively ligating one of the many feeder arterioles into the cornea, we caused network‐wide remodeling of blood flow. Ligation of the feeder vessel allowed us to observe changes in blood flow and Tie2‐GFP expression pre‐ligation, 1 hour post‐ligation, and 48 hours post‐ligation (and blood flow remodeling) in the same vessel segments.Thus far, we observed that blood flow precedes EC expression of Tie2 in an angiogenic network, with only 16.8% of a perfused vessel expressing Tie2‐GFP compared to 92.3% at Day 5 (p = 0.003). The magnitude of blood flow‐induced wall shear stress across a single vessel (calculated) is directly proportional to Tie2‐GFP expression density in the same vessel (r2 = 0.74). Also, the change in shear stress is directly proportional to a change in Tie2 expression (r2 = 0.93). We also observe PCs recruited to vessels prior to Tie2 expression along neovessels.These results establish a novel method of observing blood flow, EC‐PC interactions, and expression of a gene‐driven fluorescent reporter across an intact microvascular network in a living mammal. Further, they implicate the role of blood flow‐induced shear stress in the onset and maintenance of endothelial maturity.Support or Funding InformationSupported by the Wagner Fellowship, and NIH Grants 5 T32 HL007284‐37, HL082838‐02, and EY022063‐01.