Arterial occlusive disease (AOD) is the leading cause of morbidity and mortality through the developed world, which creates a significant need for effective therapies to halt disease progression. Despite success of animal and small-scale human therapeutic arteriogenesis studies, this promising concept for treating AOD has yielded largely disappointing results in large-scale clinical trials. One reason for this lack of successful translation is that endogenous arteriogenesis is highly dependent on a poorly understood sequence of events and interactions between bone marrow derived cells (BMCs) and vascular cells, which makes designing effective therapies difficult. We contend that the process follows a complex, ordered sequence of events with multiple, specific BMC populations recruited at specific times and locations. Here we present the evidence suggesting roles for multiple BMC populations from neutrophils and mast cells to progenitor cells and propose how and where these cell populations fit within the sequence of events during arteriogenesis. Disruptions in these various BMC populations can impair the arteriogenesis process in patterns that characterize specific patient populations. We propose that an improved understanding of how arteriogenesis functions as a system can reveal individual BMC populations and functions that can be targeted for overcoming particular impairments in collateral vessel development.

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.

Venomotion, spontaneous cyclic contractions of venules, was first observed in the bat wing 160 years ago. Of all the functional roles proposed since then, propulsion of blood by venomotion remains the most controversial. Common animal models that require anesthesia and surgery have failed to provide evidence for venular pumping of blood. To determine whether venomotion actively pumps blood in a minimally invasive, unanesthetized animal model, we reintroduced the batwing model. We evaluated the temporal and functional relationship between the venous contraction cycle and blood flow and luminal pressure. Furthermore, we determined the effect of inhibiting venomotion on blood flow. We found that the active venous contractions produced an increase in the blood flow and exhibited temporal vessel diameter-blood velocity and pressure relationships characteristic of a peristaltic pump. The presence of valves, a characteristic of reciprocating pumps, enhances the efficiency of the venular peristaltic pump by preventing retrograde flow. Instead of increasing blood flow by decreasing passive resistance, venular dilation with locally applied sodium nitroprusside decreased blood flow. Taken together, these observations provide evidence for active venular pumping of blood. Although strong venomotion may be unique to bats, venomotion has also been inferred from venous pressure oscillations in other animal models. The conventional paradigm of microvascular pressure and flow regulation assumes venules only act as passive resistors, a proposition that must be reevaluated in the presence of significant venomotion.

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.