Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal α-Adrenergic Signaling and a Transient Loss of Vascular Tone.

Stimulating collateral arteriogenesis is an attractive therapeutic target for peripheral artery disease (PAD). However, the potency of arteriogenesis-stimulation in animal models has not been matched with efficacy in clinical trials. This may be because the presence of enlarged collaterals is not sufficient to relieve symptoms of PAD, suggesting that collateral function is also important. Specifically, collaterals are the primary site of vascular resistance following arterial occlusion, and impaired collateral vasodilation could impact downstream tissue perfusion and limb function. Therefore, we evaluated the effects of arteriogenesis on collateral vascular reactivity. Following femoral artery ligation in the mouse hindlimb, collateral functional vasodilation was impaired at day 7 (17 ± 3 vs. 60 ± 8%) but restored by day 28. This impairment was due to a high resting diameter (73 ± 4 µm at rest vs. 84 ± 3 µm dilated), which does not appear to be a beneficial effect of arteriogenesis because increasing tissue metabolic demand through voluntary exercise decreased resting diameter and restored vascular reactivity at day 7. The high diameter in sedentary animals was not due to sustained NO-dependent vasodilation or defective myogenic constriction, as there were no differences between the enlarged and native collaterals in response to eNOS inhibition with L-NAME or L-type calcium channel inhibition with nifedipine, respectively. Surprisingly, in the context of reduced vascular tone, vasoconstriction in response to the α-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (-62 ± 2 vs. -37 ± 2%) while vasodilation in response to the α-adrenergic antagonist prazosin was reduced (6 ± 4% vs. 22 ± 16%), indicating a lack of α-adrenergic receptor activation by endogenous norepinephrine and suggesting a denervation of the neuroeffector junction. Staining for tyrosine hydroxylase demonstrated sympathetic denervation, with neurons occupying less area and located further from the enlarged collateral at day 7. Inversely, MMP2 presence surrounding the enlarged collateral was greater at day 7, suggesting that denervation may be related to extracellular matrix degradation during arteriogenesis. Further investigation on vascular wall maturation and the functionality of enlarged collaterals holds promise for identifying novel therapeutic targets to enhance arteriogenesis in patients with PAD.

Arterialized collateral capillaries progress from nonreactive to capable of increasing perfusion in an ischemic arteriolar tree.

OBJECTIVE: CCA, outward remodeling of capillaries that anastomose 2 arteriolar trees with different parent feed arteries, may represent a therapeutic target for patients who lack collaterals. ACCs can reperfuse an ischemic tree, but their functional capacity is unknown. Therefore, we determined whether ACCs mature into resistance vessels that regulate blood flow following arterial occlusion. METHODS: We ligated the lateral spinotrapezius feed artery in Balb/C mice, which induces CCA. At days 7 and 21 following occlusion, we measured vasodilation of ACCs using intravital microscopy and blood flow in the ischemic tree using LSF. We determined the presence of ACCs and neurovascular alignment with immunofluorescence. RESULTS: At day 7, ACCs do not vasodilate following muscle contraction and have reduced responses to endothelial- and smooth muscle-dependent agents. By day 21, ACCs exhibit normal vasodilation, accompanied by normalized increases in relative blood flow to the ischemic zone. Although functioning as resistance vessels by regulating blood flow, ACCs do not appear to be innervated. CONCLUSIONS: ACCs mature into resistance vessels that regulate blood flow to the downstream tissue. Therefore, induction of mature ACCs may be a target for reducing ischemia in patients who lack collateral networks.

Murine spinotrapezius model to assess the impact of arteriolar ligation on microvascular function and remodeling.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published(1-3). For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 µm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Chronic hindlimb ischemia impairs functional vasodilation and vascular reactivity in mouse feed arteries.

Vasodilation of lower leg arterioles is impaired in animal models of chronic peripheral ischemia. In addition to arterioles, feed arteries are a critical component of the vascular resistance network, accounting for as much as 50% of the pressure drop across the arterial circulation. Despite the critical importance of feed arteries in blood flow control, the impact of ischemia on feed artery vascular reactivity is unknown. At 14 days following unilateral resection of the femoral-saphenous artery-vein pair, functional vasodilation of the profunda femoris artery was severely impaired, 11 ± 9 versus 152 ± 22%. Although endothelial and smooth muscle-dependent vasodilation were both impaired in ischemic arteries compared to control arteries (Ach: 40 ± 14 versus 81 ± 11%, SNP: 43 ± 12 versus and 85 ± 11%), the responses to acetylcholine and sodium nitroprusside were similar, implicating impaired smooth muscle-dependent vasodilation. Conversely, vasoconstriction responses to norepinephrine were not different between ischemic and control arteries, -68 ± 3 versus -66 ± 3%, indicating that smooth muscle cells were functional following the ischemic insult. Finally, maximal dilation responses to acetylcholine, ex vivo, were significantly impaired in the ischemic artery compared to control, 71 ± 9 versus 97 ± 2%, despite a similar generation of myogenic tone to the same intravascular pressure (80 mmHg). These data indicate that ischemia impairs feed artery vasodilation by impairing the responsiveness of the vascular wall to vasodilating stimuli. Future studies to examine the mechanistic basis for the impact of ischemia on vascular reactivity or treatment strategies to improve vascular reactivity following ischemia could provide the foundation for an alternative therapeutic paradigm for peripheral arterial occlusive disease.

Increased hexokinase II expression in the renal glomerulus of mice in response to arsenic.

Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

A modified fluorescent microsphere-based approach for determining resting and hyperemic blood flows in individual murine skeletal muscles.

The goal of this study was to develop a modified fluorescent microsphere-based approach for measuring resting and hyperemic blood flows in individual mouse skeletal muscles. Absolute resting blood flow in the left gracilis posterior was 1.04+/-0.12 ml x min(-1).g(-1), while functional hyperemia following muscle activity was 5.94+/-1.33 ml x min(-1) x g(-1). Measuring absolute blood flow requires sampling arterial blood that serves as a flow-rate and concentration reference to the fluorescent microsphere (FMS) content in the tissue-of-interest for calculating the flow value. Because sampling arterial blood can impair cardiovascular function in the mouse, we also modified our FMS approach to determine relative blood flows in the left gracilis posterior by using the contralateral muscle as our reference in blood flow calculations. Absolute and relative hyperemia measurements detect similar increases in blood flow - 521.93+/-216.76% and 555.24+/-213.82%, respectively. However, sampling arterial blood during absolute blood flow measurements significantly decreased mean arterial pressure from the beginning to the end of our experiments, from 102.7+/-2.18 to 75.5+/-9.71 mm Hg. This decrease was not seen when measuring relative blood flows. This approach provides critical advantages over contemporary blood flow measurement approaches by allowing blood flow measurements in small and non-superficial tissues.