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. 2015 Nov;35(11):2354-65.
doi: 10.1161/ATVBAHA.115.305775. Epub 2015 Sep 3.

Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction

Free PMC article

Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction

Joshua L Heuslein et al. Arterioscler Thromb Vasc Biol. .
Free PMC article

Erratum in

  • Correction.
    Arterioscler Thromb Vasc Biol. 2015 Nov;35(11):e58. doi: 10.1161/ATV.0000000000000025. Arterioscler Thromb Vasc Biol. 2015. PMID: 26490278


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.

Approach and 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.

Keywords: endothelial cells; femoral artery; gene expression; hemodynamics; peripheral arterial disease.


Fig. 1
Fig. 1. Collateral artery endothelial cells repolarize in response to shear stress reversal after FAL
A) Schematic of the primary gracilis adductor collateral flow pathways after femoral arterial ligation (FAL). The femoral artery is ligated just distal to the epigastric artery thereby creating a change in flow direction at the saphenous branch entrance region. Arrows indicate the direction and magnitude of blood flow pre- (yellow) and post- (white) FAL. Flow magnitude increases in both the muscular branch and saphenous entrance regions, while flow direction also changes in the saphenous region. B) Representative images of collateral regions immunolabeled for the Golgi apparatus, the position of which was used to determine planar polarization, a marker of endothelial response to flow direction. Images are oriented such that the muscular branch is toward the left and the saphenous artery is toward the right. Arrows indicate blood flow direction. Note the upstream position of the Golgi apparatus (anti-giantin, outlined in green) with respect to the nucleus (Draq5, outline in magenta) (Scale bar=25 μm). C) Diagram depicting the method used to quantify of polarization. The position of the Golgi apparatus (green) with respect to the nucleus (magenta) was used to classify EC polarization as toward either the saphenous (black bars) or muscular branch (white bars) arteries. D) Bar graph of pre-FAL EC planar polarization. ECs in the entrance regions show upstream polarization, while those central region show no directional bias (n=4). E) Bar graph of 24 hour post-FAL EC planar polarization. ECs in all regions show upstream polarization toward the muscular branch entrance region (n=4). *p<0.05 between muscular and saphenous polarization within the given region. Data are mean ± SEM.
Fig. 2
Fig. 2. Gracilis collateral artery regions exposed to flow-reversal exhibit enhanced arteriogenesis after FAL
A) Representative vascular cast images of gracilis collateral arteries from unligated sham control (top) and FAL-treated (bottom) mice. Arrows indicate the direction and relative magnitude of blood flow pre- (yellow) and post- (white) FAL. Dashed lines indicate the collateral artery region (muscular=blue; saphenous=orange) and approximate location where muscles were cut for regional cross-sectional analysis. Boxes indicate location at which lumenal diameters were measured (i.e. near the first transverse arteriole branch point). B) Bar graph of collateral artery lumenal diameter at both the non-reversed (muscular branch, blue) and flow-reversed (saphenous, orange) entrance regions. (n=6–7 mice per day). C) Representative H&E stained cross-sections of gracilis muscles. Microfil casting material (M) is evident in artery lumens. D–F) Bar graphs of total cross sectional lumenal diameter, wall area, and wall thickness (n=6–7 mice per day). *p<0.05 between ligated and unligated within the given region at the specified time-point; †p<0.05 between regions (i.e. saphenous vs. muscular) within the given treatment (i.e. ligated or unligated) at the specified time-point. Data are mean ± SEM.
Fig. 3
Fig. 3. Genome-wide analysis indicates that flow reversal broadly enhances the arteriogenic transcriptional profile
A) Schematic depicting shear stress conditions applied to HUVECs to simulate reversing (saphenous) and non-reversing (muscular) regions. HUVECs were preconditioned for 24 hours at 15 dynes/cm2. Shear stress was then increased (or kept constant in the Control group) to 30 dynes/cm2 in the same or opposite direction. B) Venn diagram of Affymetrix ST 1.0 human cDNA array data (n=4) comparing significantly altered transcripts (FDR<0.1) in non-reversed vs. control (NvC) conditions to those significantly altered with reversed direction vs. control (RvC). Red text and arrows=upregulated; Blue text and arrows=downregulated. C) Scatterplot showing gene expression changes between RvC and NvC conditions. While a similar overall pattern of expression (i.e. either up in both conditions or down in both conditions) was observed, gene expression was broadly amplified with the reversed shear stress condition (red region=greater up-regulation in RvC; blue region=greater down-regulation in RvC; black line=equal RvC and NvC). D–G) Heatmaps grouped along significantly altered canonical pathways (cell-cell junctional signaling, protein kinase A signaling, MAP kinase signaling, and phosphodiesterase signaling) activated in both RvC and NvC conditions (see Table S3A). Similar patterns of activation were observed between the conditions, but to a greater degree in the reversed flow condition.
Fig. 4
Fig. 4. Flow reversal enhances monocyte adhesion in-vitro in an ICAM-1 dependent manner
A) NFκB activity (n=3) relative to control in HUVECs exposed to reversing (R, black) and non-reversing (N, white) shear conditions 1 hour post-“FAL” in vitro (Figure 3A). B, C) ICAM-1 mRNA expression by RT-PCR (n=8) and protein expression (n=4) in HUVECs relative to control 6 hours post in vitro “FAL”. D) Bar graph quantifying relative number of adhered monocytes in each condition E) Representative FOVs of THP-1 monocytes adhered to HUVECs exposed to reversed and non-reversed flow conditions transfected with either ICAM-1 siRNA (siICAM-1) or non-targeting control (siC) (Scale bar=100μm). †p<0.05 between reversing (R) and non-reversing (N);*p<0.05 between siC and siICAM-1 for given flow condition. Data are mean ± SEM.
Fig. 5
Fig. 5. ICAM-1 is necessary for enhanced macrophage recruitment and amplified arteriogenesis in collateral segments exposed to a flow reversal
A) Representative cross-sections of gracilis collateral artery regions in WT C57BL/6 and ICAM-1−/− mice 3 days post-FAL immunolabeled for macrophage marker, Mac3 (green) and smooth muscle alpha actin (red). Dotted line indicates pericollateral region (25μm from vessel wall) used for quantification. Arrows indicate Mac3+ cells. (Scale bar=50μm) B) Bar graph of pericollateral Mac3+ cells. (n=4–5). C) Representative vascular cast images from ICAM−/− mice 14 days post-FAL from both muscular and saphenous regions. (Scale bar=100μm). Arrows indicate the measured collateral artery while arrowheads indicate transverse arterioles. D) Bar graph of regional lumenal diameter for both WT and ICAM-1−/− mice (n=6). *p<0.05 between ligated and unligated within the given region; †p<0.05 between regions (saphenous versus muscular) within the given treatment (ligated or unligated). Data are mean ± SEM.

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