Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction.
OBJECTIVE
Collateral arteriogenesis, the growth of existing arterial vessels to a larger diameter, is a fundamental adaptive response that is often critical for the perfusion and survival of tissues downstream of chronic arterial occlusion(s). Shear stress regulates arteriogenesis; however, the arteriogenic significance of reversed flow direction, occurring in numerous collateral artery segments after femoral artery ligation, is unknown. Our objective was to determine if reversed flow direction in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis.


RESULTS
Collateral segments experiencing reversed flow direction after femoral artery ligation in C57BL/6 mice exhibit increased pericollateral macrophage recruitment, amplified arteriogenesis (30% diameter and 2.8-fold conductance increases), and remarkably permanent (12 weeks post femoral artery ligation) remodeling. Genome-wide transcriptional analyses on human umbilical vein endothelial cells exposed to reversed flow conditions mimicking those occurring in vivo yielded 10-fold more significantly regulated transcripts, as well as enhanced activation of upstream regulators (nuclear factor κB [NFκB], vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-β) and arteriogenic canonical pathways (protein kinase A, phosphodiesterase, and mitogen-activated protein kinase). Augmented expression of key proarteriogenic molecules (Kruppel-like factor 2 [KLF2], intercellular adhesion molecule 1, and endothelial nitric oxide synthase) was also verified by quantitative real-time polymerase chain reaction, leading us to test whether intercellular adhesion molecule 1 or endothelial nitric oxide synthase regulate amplified arteriogenesis in flow-reversed collateral segments in vivo. Interestingly, enhanced pericollateral macrophage recruitment and amplified arteriogenesis was attenuated in flow-reversed collateral segments after femoral artery ligation in intercellular adhesion molecule 1(-/-) mice; however, endothelial nitric oxide synthase(-/-) mice showed no such differences.


CONCLUSIONS
Reversed flow leads to a broad amplification of proarteriogenic endothelial signaling and a sustained intercellular adhesion molecule 1-dependent augmentation of arteriogenesis. Further investigation of the endothelial mechanotransduction pathways activated by reversed flow may lead to more effective and durable therapeutic options for arterial occlusive diseases.
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Joshua L
JL
Heuslein
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Joshua K
JK
Meisner
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Xuanyue
X
Li
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Ji
J
Song
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Helena
H
Vincentelli
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Ryan J
RJ
Leiphart
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Elizabeth G
EG
Ames
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.).
Brett R
BR
Blackman
Brett R
BR
Blackman
From the Departments of Biomedical Engineering (J.L.H., J.K.M., X.L., J.S., H.V., R.J.L., E.G.A., R.J.P.), Molecular Physiology and Biological Physics (E.G.A.), Radiology (R.J.P.), and Radiation Oncology (R.J.P.), University of Virginia, Charlottesville; and HemoShear Therapeutics LLC, Charlottesville, VA (B.R.B.). rprice@virginia.edu.
Richard J
RJ
Price
D000818:Animals; D001158:Arteries / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D001783:Blood Flow Velocity; D002478:Cells, Cultured; D003097:Collateral Circulation; D004195:Disease Models, Animal; D005263:Femoral Artery / Q000503:physiopathology / Q000601:surgery; D005786:Gene Expression Regulation; D006614:Hindlimb; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D008026:Ligation; D008264:Macrophages / Q000378:metabolism; D008297:Male; D040542:Mechanotransduction, Cellular; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D012039:Regional Blood Flow; D013314:Stress, Mechanical; D013997:Time Factors; D066253:Vascular Remodeling
C573420:ICAM1 protein, human; C090509:Icam1 protein, mouse; D016328:NF-kappa B; D018799:Intercellular Adhesion Molecule-1; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse
endothelial cells; femoral artery; gene expression; hemodynamics; peripheral arterial disease