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Microsurgical Induced Angiogenesis
Patrick C. Hancock, MD, Mingjie Sun, MD, Srinivas V. Koduru, PhD, Dino J. Ravnic, DO, MPH, MSc.
The Pennsylvania State University College of Medicine, Hershey, PA, USA.

PURPOSE: Blood vessel growth is essential for the viability of all tissues. Angiogenesis is paramount to normal development, injury recovery, and more recently, vital to the integration of bioengineered grafts. However, innate blood vessel proliferation is slow and often inadequate. Therapeutic angiogenesis aims to stimulate the growth of new blood vessels from preexisting vasculature using various strategies such as exogenous pharmacologic stimulation, cytokine supplementation, stem cells, and in situ cell homing. However, widespread clinical translation has proven suboptimal and the side-effects significant. Here we describe a novel microsurgical intervention to safely induce rapid therapeutic angiogenesis, which could be used in a variety of settings.
METHODS: The rat hindlimb macrovasculature (N=10) was micropunctured (MP) using a 60 μm diameter needle at defined intervals prior to implantation of a prepared Type I collagen scaffold directly on top of the punctured segment. The contralateral hindlimb served as a normal (non-micropunctured) intrinsic control. Collagen grafts and the underlying vascular segment were explanted en bloc for gross and histologic analysis after 24, 48, and 72 hours. Cellular infiltration and capillary ingrowth were assessed per high powered field (HPF) following trichrome staining. Single cell-lined lumens were characterized by both number and diameter; from which the overall perfusable area was calculated. The presence of endothelial cells was verified by CD31/PECAM-1, von Willebrand factor (vWF), and Tie2 immunofluorescence staining. Endothelial cell proliferation was estimated by VEGFR2, Notch 1, and delta-like 4 (DLL4). Statistical significance was determined using the one-tailed Mann-Whitney U test.
RESULTS: Micropunctured vessels demonstrated an increase in cellular infiltration, capillary outgrowth and luminal diameter across all time points when compared to non-micropunctured samples. This resulted in a two-fold overall increase of perfusable area within the explanted collagen, reaching statistical significance (P<0.05) at 24 and 72 hours (Table 1). Immunofluorescence labeling of CD31, vWF, and Tie2 confirmed the increased presence of endothelial cells along the periphery of measured luminal structures in micropunctured specimens. Additionally, micropunctured samples exhibited increased cellular staining of VEGFR2, Notch 1, and DLL4 (Table 2). This suggests the specific proliferation of endothelial stalk and tip cells which are fundamental to sprouting angiogenesis.
CONCLUSION: Microsurgical induction of angiogenesis was first described with the arduous creation of arteriovenous-loops. However, the time to new capillary formation is slow, limiting widespread clinical translation. Here we describe an innovative alternative technique that leads to the rapid proliferation of endothelial cells and robust capillary formation. Because our approach evaluates the paradigm of therapeutic angiogenesis from a completely novel perspective it has enormous potential to mitigate ischemia in both conventional surgery and bioengineered applications.

Table 1. Collagen Graft Vascularization. Results per high powered field shown as mean ▒ SD.
24 hours48 hours72 hours
% area cellular infiltrationControl6.0 ▒ 3.812.44 ▒ 7.8022.81 ▒ 10.35
Micropuncture11.6 ▒ 5.7818.77 ▒ 8.0235.04 ▒ 10.42
Number of new vesselsControl3.20 ▒ 1.975.34 ▒ 2.4110.93 ▒ 3.22
Micropuncture5.07 ▒ 4.458.80 ▒ 5.1213.07 ▒ 5.86
Diameter of new vessels (Ám)Control14.01 ▒ 6.1417.03 ▒ 11.318.05 ▒ 9.08
Micropuncture17.15 ▒ 8.7019.64 ▒ 11.4723.36 ▒ 12.81
Perfusable area (Ám2)Control527.7 ▒ 5181465 ▒ 12142995 ▒ 1484
Micropuncture1288 ▒ 12872853 ▒ 28125897 ▒ 3271
P value0.0480.0790.005

Table 2. Cellular Markers of Angiogenesis. Results per high powered field shown as mean ▒ SD.
24 hours48 hours72 hours
vWFControl16.0 ▒ 4.221.2 ▒ 10.939.4 ▒ 13.4
Micropuncture25.2 ▒ 9.042.6 ▒ 25.368.4 ▒ 37.2
Tie2Control16.0 ▒ 3.520.8 ▒ 4.841.4 ▒ 21.7
Micropuncture20.4 ▒ 5.042.6 ▒ 13.180.2 ▒ 31.1
Notch 1Control13.0 ▒ 2.218.6 ▒ 3.840.4 ▒ 22.3
Micropuncture18.6 ▒ 1.341.2 ▒ 14.479.0 ▒ 29.1
DLL4Control19.8 ▒ 10.926.0 ▒ 12.949.0 ▒ 18.5
Micropuncture20.2 ▒ 7.840.8 ▒ 15.0100.4 ▒ 43.0

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