Objective Chronic arterial occlusion results in arteriogenesis of collateral blood vessels. This process has been shown to be dependent upon the recruitment of growth-promoting macrophages to remodeling collaterals. However, the potential role of venules in monocyte recruitment during microvascular arteriogenesis is not well demonstrated. First, we aim to document that arteriogenesis occurs in the mouse spinotrapezius ligation model. Then, we investigate the temporal and spatial distribution, as well as proliferation, of monocytes/macrophages recruited to collateral arterioles in response to elevated fluid shear stress. Approach and Results Laser speckle flowmetry confirmed a post-ligation increase in blood velocity within collateral arterioles but not venules. After 72 hours post-ligation, collateral arteriole diameters were increased, proliferating cells were identified in vessel walls of shear-activated collaterals, and perivascular CD206+ macrophages demonstrated proliferation. An EdU assay identified proliferation. CD68+CD206+ cells around collaterals were increased 96%, while CX3CR1(+/GFP ) cells were increased 126% in ligated versus sham groups after 72 hours. CX3CR1(+/GFP ) cells were predominately venule-associated at 6 hours post-ligation; and CX3CR1(+/GFP hi) cells shifted from venule-associated to arteriole-associated between 6 and 72 hours post-surgery exclusively in ligated muscle. We report accumulation and extravasation of adhered CX3CR1(+/GFP) cells in and from venules, but not arterioles, following ligation. Conclusions Our results demonstrate that arteriogenesis occurs in the murine spinotrapezius ligation model and implicate post-capillary venules as the site of tissue entry for circulating monocytes. Local proliferation of macrophages also is documented. These data open up questions concerning the role of arteriole-venule communication during monocyte recruitment.

The growth of endogenous collateral arteries that bypass arterial occlusion(s), or arteriogenesis, is a fundamental shear stress-induced adaptation with implications for treating peripheral arterial disease. MicroRNAs (miRs) are key regulators of gene expression in response to injury and have strong therapeutic potential. In a previous study, we identified miR-146a as a candidate regulator of vascular remodeling. Here, we tested whether miR-146a regulates in vitro angiogenic endothelial cell (EC) behaviors, as well as perfusion recovery, arteriogenesis, and angiogenesis in response to femoral arterial ligation (FAL) in vivo. We found miR-146a inhibition impaired EC tube formation and migration in vitro. Following FAL, Balb/c mice were treated with a single, intramuscular injection of anti-miR-146a or scramble locked nucleic acid (LNA) oligonucleotides directly into the non-ischemic gracilis muscles. Serial laser Doppler imaging demonstrated that anti-miR-146a treated mice exhibited significantly greater perfusion recovery (a 16% increase) compared mice treated with scramble LNA. Moreover, anti-miR-146a treated mice exhibited a 22% increase in collateral artery diameter compared to controls, while there was no significant effect on in vivo angiogenesis or muscle regeneration. Despite exerting no beneficial effects on angiogenesis, the inhibition of mechanosensitive miR-146a enhances perfusion recovery after FAL via enhanced arteriogenesis.

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 flow direction reversal, occurring in numerous collateral artery segments after femoral artery ligation (FAL), is unknown. Our objective was to determine if flow direction reversal in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis. Approach and Results Collateral segments experiencing flow reversal after FAL 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-FAL) remodeling. Genome-wide transcriptional analyses on HUVECs exposed to flow reversal conditions mimicking those occurring in-vivo yielded 10-fold more significantly regulated transcripts, as well as enhanced activation of upstream regulators (NFκB, VEGF, FGF2, TGFβ) and arteriogenic canonical pathways (PKA, PDE, MAPK). Augmented expression of key pro-arteriogenic molecules (KLF2, ICAM-1, eNOS) was also verified by qRT-PCR, leading us to test whether ICAM-1 and/or eNOS 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 FAL in ICAM-1−/− mice; however, eNOS−/− mice showed no such differences. Conclusions Flow reversal leads to a broad amplification of pro-arteriogenic endothelial signaling and a sustained ICAM-1-dependent augmentation of arteriogenesis. Further investigation of the endothelial mechanotransduction pathways activated by flow reversal may lead to more effective and durable therapeutic options for arterial occlusive diseases.

In this study, we explored whether topical application of antibodies targeting tumor necrosis factor‐α (TNF‐α) or interleukin‐6 (IL‐6) conjugated to hyaluronic acid (HA) could reduce the extension of necrosis by modulating inflammation locally in a partial‐thickness rat burn model. Partial‐thickness to deep partial‐thickness burn injuries present significant challenges in healing, as these burns often progress following the initial thermal insult, resulting in necrotic expansion and increased likelihood of secondary complications. Necrotic expansion is driven by a microenvironment with elevated levels of pro‐inflammatory mediators, and local neutralization of these using antibody conjugates could reduce burn progression. Trichrome‐stained tissue sections indicated the least necrotic tissue in (anti‐TNF‐α)‐HA‐treated sites, while (anti‐IL‐6)‐HA‐treated sites displayed similar outcomes to saline controls. This was confirmed by vimentin immunostaining, which demonstrated that HA treatment alone reduced burn progression by nearly 30%, but (anti‐TNF‐α)‐HA reduced it by approximately 70%. At all time points, (anti‐TNF‐α)‐HA‐treated sites showed reduced tissue levels of IL‐1β compared to controls, suggesting inhibition of a downstream mediator of inflammation. Decreased macrophage infiltration in (anti‐TNF‐α)‐HA‐treated sites compared to controls was elucidated by immunohistochemical staining of macrophages, suggesting a reduction in overall inflammation in all time points. These results suggest that local targeting of TNF‐α may be an effective strategy for preventing progression of partial‐thickness burns.

BackgroundArteriogenesis is initiated by increased shear stress and is thought to continue until shear stress is returned to its original “set point.” However, the molecular mechanism(s) through which shear stress set point is established by endothelial cells (ECs) are largely unstudied. Here, we tested the hypothesis that DNA methyltransferase 1 (DNMT1)–dependent EC DNA methylation affects arteriogenic capacity via adjustments to shear stress set point.Methods and ResultsIn femoral artery ligation–operated C57BL/6 mice, collateral artery segments exposed to increased shear stress without a change in flow direction (ie, nonreversed flow) exhibited global DNA hypermethylation (increased 5‐methylcytosine staining intensity) and constrained arteriogenesis (30% less diameter growth) when compared with segments exposed to both an increase in shear stress and reversed‐flow direction. In vitro, ECs exposed to a flow waveform biomimetic of nonreversed collateral segments in vivo exhibited a 40% increase in DNMT1 expression, genome‐wide hypermethylation of gene promoters, and a DNMT1‐dependent 60% reduction in proarteriogenic monocyte adhesion compared with ECs exposed to a biomimetic reversed‐flow waveform. These results led us to test whether DNMT1 regulates arteriogenic capacity in vivo. In femoral artery ligation–operated mice, DNMT1 inhibition rescued arteriogenic capacity and returned shear stress back to its original set point in nonreversed collateral segments.ConclusionsIncreased shear stress without a change in flow direction initiates arteriogenic growth; however, it also elicits DNMT1‐dependent EC DNA hypermethylation. In turn, this diminishes mechanosensing, augments shear stress set point, and constrains the ultimate arteriogenic capacity of the vessel. This epigenetic effect could impact both endogenous collateralization and treatment of arterial occlusive diseases.

Objective To estimate the relative influence of input pressure and arteriole rarefaction on gastrocnemius muscle perfusion in patients with peripheral arterial disease (PAD) after exercise and/or percutaneous interventions. Methods A computational network model of the gastrocnemius muscle microcirculation was adapted to reflect rarefaction based on arteriolar density measurements from PAD patients, with and without exercise. A normalized input pressure was applied at the feeder artery to simulate both reduced and restored ankle-brachial index (ABI) in the PAD condition. Results In simulations of arteriolar rarefaction, resistance increased non-linearly with rarefaction, leading to a disproportionally large drop in perfusion. Additionally, perfusion was less sensitive to changes in input pressure as the degree of rarefaction increased. Reduced arteriolar density was observed in PAD patients and improved 33.8% after 3 months of exercise. In model simulations of PAD, ABI restoration yielded perfusion recovery to only 66% of baseline. When exercise training was simulated by reducing rarefaction, ABI restoration increased perfusion to 80% of baseline. Conclusion Microvascular resistance increases non-linearly with increasing arteriole rarefaction. Therefore, muscle perfusion becomes disproportionally less sensitive to ABI restoration as arteriole rarefaction increases. These results highlight the importance of restoring both microvascular structure and upstream input pressure in PAD therapy.

Arteriogenesis, the growth of endogenous collateral arteries bypassing arterial occlusion(s), is a fundamental shear stress-induced adaptation with implications for treating peripheral arterial disease (PAD). Nonetheless, endothelial mechano-signaling during arteriogenesis is incompletely understood. Here we tested the hypothesis that a mechanosensitive microRNA, miR-199a-5p, regulates perfusion recovery and collateral arteriogenesis following femoral arterial ligation (FAL) via control of monocyte recruitment and pro-arteriogenic gene expression. We have previously shown that collateral artery segments exhibit distinctly amplified arteriogenesis if they are exposed to reversed flow following FAL in the mouse. We performed a genome-wide analysis of endothelial cells exposed to a biomimetic reversed flow waveform. From this analysis, we identified mechanosensitive miR-199a-5p as a novel candidate regulator of collateral arteriogenesis. In vitro, miR-199a-5p inhibited pro-arteriogenic gene expression (IKKβ, Cav1) and monocyte adhesion to endothelium. In vivo, following FAL in mice, miR-199a-5p overexpression impaired foot perfusion and arteriogenesis. In contrast, a single intramuscular anti-miR-199a-5p injection elicited a robust therapeutic response, including complete foot perfusion recovery, markedly augmented arteriogenesis (>3.4-fold increase in segment conductance), and improved gastrocnemius tissue composition. Finally, we found plasma miR-199a-5p to be elevated in human PAD patients with intermittent claudication compared to a risk factor control population. Through our transformative analysis of endothelial mechano-signaling in response to a biomimetic amplified arteriogenesis flow waveform, we have identified miR-199a-5p as both a potent regulator of arteriogenesis and a putative target for treating PAD.

Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.