Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal alpha-Adrenergic Signaling and a Transient Loss of Vascular Tone.
Blockage of thigh arteries can lead to loss of blood flow to the lower leg and possibly amputation if the tissue downstream of the blockage dies- this is the leading cause of amputation in the Unites States. Unfortunately, angioplasty and bypass surgery are not as effective in the leg as they are in the heart, benefiting only half of the patients with the worst blockages. Those patients who are not helped by angioplasty or bypass surgery then have a very high risk of death – a 20% 5-year survival rate, which is similar to pancreatic cancer. Therefore, researchers are investigating alternative approaches to maintain blood flow to the lower leg in patients whose thigh arteries are blocked. One of the most common approaches is to stimulate growth of natural bypasses. Mammals have natural bypasses that can re-route blood flow around the blocked limb arteries, if they are able to enlarge and function effectively. Many patients do not have enlarged and functional natural bypasses, so stimulating the enlargement of these blood vessels is a potential target for new therapies. Unfortunately, despite very promising results in laboratory animals, numerous clinical trials for therapies have failed to stimulate natural bypass enlargement. We think that one reason for this failure is an insufficient understanding of the process that natural bypasses go through to enlarge. Specifically, we think that it is important for natural bypasses to not only grow, but also be able to function by regulating blood flow. Normally, arteries change their diameter on a moment-to-moment basis through the contraction and relaxation of the muscle cells that surround them- an involuntary process, unlike our voluntary muscles that allow our bodies to move; also, this moment-to-moment change is different from enlargement, which involves the artery cells dividing to make a structurally larger blood vessel. For example, after a meal, muscle cells in blood vessels of the digestive system will relax to increase the diameter and allow more blood flow to feed the stomach as it grinds up our swallowed food, whereas when we are cold, muscle cells in the blood vessels of our skin will contract to decrease the diameter and divert blood flow to the core. We hypothesized that when natural bypasses enlarge, that the muscle cells surrounding them will lose their ability to contract and relax normally. We supported this hypothesis in laboratory mice and determined that the muscle cells of enlarged natural bypasses are initially unable contract, but regain this ability after several weeks. We also determined that the reason for this inability to contract is that the muscle cells of the enlarged natural bypasses lose their innervation from involuntary nerves that normally control contraction and relaxation. We expect that considering both the growth (i.e. enlargement) and function (i.e. ability to contract and relax) of natural bypasses will help in the development of new therapies that effectively restore blood flow in patients whose thigh arteries are blocked.
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 mum at rest vs. 84 +/- 3 mum 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 alpha-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (-62 +/- 2 vs. -37 +/- 2%) while vasodilation in response to the alpha-adrenergic antagonist prazosin was reduced (6 +/- 4% vs. 22 +/- 16%), indicating a lack of alpha-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.