P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia.

OBJECTIVE: Arteriogenesis represents the maturation of preformed vascular connections in response to flow changes and shear stress. These collateral vessels can restore up to 60% of the native blood flow. Shear stress and vascular injury can induce the release of nucleotides from vascular smooth muscle cells and platelets that can serve as signaling ligands, suggesting they may be involved in mediating arteriogenesis. The P2Y2 nucleotide receptor (P2Y2R) has also been shown to mediate smooth muscle migration and arterial remodeling. Thus, we hypothesize that P2Y2R mediates arteriogenesis in response to ischemia. METHODS: Hind limb ischemia was induced by femoral artery ligation (FAL) in C57Bl/6NJ or P2Y2R negative mice (P2Y2(-/-)). Hind limb perfusion was measured with laser Doppler perfusion imaging and compared with the sham-operated contralateral limb immediately and at 3, 7, 14, 21, and 28 days after ligation. Collateral vessel size was measured by Microfil casting. Muscle specimens were harvested and analyzed with immunohistochemistry for Ki67, vascular cell adhesion molecule, macrophages, and muscle viability by hematoxylin and essoin stain. RESULTS: Hind limb ischemia induced by FAL in C57Bl/6NJ mice resulted in significant ischemia as measured by laser Doppler perfusion imaging. There was rapid recovery to nearly normal levels of perfusion by 2 weeks. FAL in P2Y2(-/-) mice resulted in severe ischemia with greater tissue loss. Recovery of perfusion was impaired, achieving only 40% compared with wild-type mice by 28 days. Collateral vessels in the P2Y2(-/-) mice were underdeveloped, with reduced vascular cell proliferation and smaller vessel size. The collaterals were ∼65% the size of wild-type collateral vessels (P = .011). Angiogenesis at 28 days in the ischemic muscle, however, was greater in the P2Y2(-/-) mice (P < .001), possibly related to persistent ischemia leading and angiogenic drive. Early macrophage recruitment was reduced by nearly 70% in P2Y2(-/-) despite significantly more myocyte necrosis. However, inflammation was greater at 28 days in the P2Y2(-/-) mice. CONCLUSIONS: P2Y2R deficiency does not alter baseline collateral vessel formation but does significantly impair collateral maturation, with resultant persistent limb ischemia despite enhanced angiogenesis. These findings reinforce the importance of arteriogenesis in the recovery of perfusion in ischemic tissues compared with angiogenesis. They also support the role of P2Y2R in mediating this process. The mechanism by which P2Y2R mediates arteriogenesis may involve the recruitment of inflammatory cells to the ischemic tissues, which is essential to arteriogenesis. Approaches to target P2Y2R may yield new therapeutic strategies for the treatment of arterial occlusive disease.

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing.

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.

Fibrosis resolution in the mouse liver: Role of Mmp12 and potential role of calpain 1/2.

Although most work has focused on resolution of collagen ECM, fibrosis resolution involves changes to several ECM proteins. The purpose of the current study was twofold: 1) to examine the role of MMP12 and elastin; and 2) to investigate the changes in degraded proteins in plasma (i.e., the "degradome") in a preclinical model of fibrosis resolution. Fibrosis was induced by 4 weeks carbon tetrachloride (CCl4) exposure, and recovery was monitored for an additional 4 weeks. Some mice were treated with daily MMP12 inhibitor (MMP408) during the resolution phase. Liver injury and fibrosis was monitored by clinical chemistry, histology and gene expression. The release of degraded ECM peptides in the plasma was analyzed using by 1D-LC-MS/MS, coupled with PEAKS Studio (v10) peptide identification. Hepatic fibrosis and liver injury rapidly resolved in this mouse model. However, some collagen fibrils were still present 28d after cessation of CCl4. Despite this persistent collagen presence, expression of canonical markers of fibrosis were also normalized. The inhibition of MMP12 dramatically delayed fibrosis resolution under these conditions. LC-MS/MS analysis identified that several proteins were being degraded even at late stages of fibrosis resolution. Calpains 1/2 were identified as potential new proteases involved in fibrosis resolution. CONCLUSION. The results of this study indicate that remodeling of the liver during recovery from fibrosis is a complex and highly coordinated process that extends well beyond the degradation of the collagenous scar. These results also indicate that analysis of the plasma degradome may yield new insight into the mechanisms of fibrosis recovery, and by extension, new "theragnostic" targets. Lastly, a novel potential role for calpain activation in the degradation and turnover of proteins was identified.

Survey of the extracellular matrix architecture across the rat arterial tree.

OBJECTIVE: To understand arterial remodeling and the pathophysiology of arterial diseases, it is necessary to understand the baseline qualities and variations in arterial structure. Arteries could differ in wall thickness, laminar structure, and laminar fenestration depending on their position within the arterial tree. We endeavored to evaluate and compare the extracellular matrix structure of different arteries throughout the arterial tree, from the aorta to the adductor muscle arteriole, with a particular focus on the internal elastic lamina (IEL). METHODS: Arterial segments were harvested from male Sprague-Dawley rats and imaged using multiple modalities. En face scans by multiphoton microscopy were used to compare native-state adventitial collagen undulation and IEL fenestration. RESULTS: Collagen undulation was similar across most examined arteries but straighter in the skeletal muscle arterioles (P < .05). The elastic lamellae showed several differences. The IEL fenestrae were similar in average size among abdominal aorta and celiac, renal, common iliac, and common femoral arteries (range, 14-24 µm2), with wide within-vessel variance (square of the standard deviation, 462-1904 µm4). However, they tended to be smaller (9.08 µm2) and less variable (square of the standard deviation, 88.3 µm4) in the popliteal artery. Fenestrae were greater in number in the superior mesenteric artery (SMA; 6686/mm2; P < .05) and profunda femoris artery (PFA; 11,042/mm2; P < .05) compared with the other examined vessels, which ranged in surface density from 3143/mm2 to 4362/mm2. The SMA and PFA also showed greater total fenestration as a proportion of the IEL surface area (SMA, 15.04%; P < .05; PFA, 24.11%; P < .001) than the other examined arteries (range of means, 4.7%-9.4%). The arteriolar IEL was structurally distinct, comparable to a low-density wireframe. Other structural differences were also noted, including differences in the number of medial lamellae along the arterial tree. CONCLUSIONS: We found that vessels at different locations along the arterial tree differ in structure. The SMA, PFA, and intramuscular arterioles have fundamental differences in the extracellular matrix structure compared with other arteries. Location-specific features such as the medial lamellae number and elastic laminar structure might have relevance to physiology and confer vulnerabilities to the development of pathology.

Intraoperative ultrasound evidence of accidental simultaneous cannulation of the common carotid artery and internal jugular vein: illustrative case.

BACKGROUND: Establishing central venous access is important to provide fluid resuscitation or medications intravenously to patients. OBSERVATIONS: Although accidental cannulation of the internal carotid artery has been reported in the literature, to our knowledge this report is the first documented intraoperative ultrasound video demonstrating accidental and simultaneous common carotid artery and internal jugular cannulation during central line placement in the internal jugular vein. LESSONS: Ultrasound use minimizes accidental carotid cannulation during central line placement in the internal jugular vein. Carotid artery puncture can be managed by external application of pressure or surgical reexploration.

Elastic Laminar Reorganization Occurs with Outward Diameter Expansion during Collateral Artery Growth and Requires Lysyl Oxidase for Stabilization.

When a large artery becomes occluded, hemodynamic changes stimulate remodeling of arterial networks to form collateral arteries in a process termed arteriogenesis. However, the structural changes necessary for collateral remodeling have not been defined. We hypothesize that deconstruction of the extracellular matrix is essential to remodel smaller arteries into effective collaterals. Using multiphoton microscopy, we analyzed collagen and elastin structure in maturing collateral arteries isolated from ischemic rat hindlimbs. Collateral arteries harvested at different timepoints showed progressive diameter expansion associated with striking rearrangement of internal elastic lamina (IEL) into a loose fibrous mesh, a pattern persisting at 8 weeks. Despite a 2.5-fold increase in luminal diameter, total elastin content remained unchanged in collaterals compared with control arteries. Among the collateral midzones, baseline elastic fiber content was low. Outward remodeling of these vessels with a 10-20 fold diameter increase was associated with fractures of the elastic fibers and evidence of increased wall tension, as demonstrated by the straightening of the adventitial collagen. Inhibition of lysyl oxidase (LOX) function with ß-aminopropionitrile resulted in severe fragmentation or complete loss of continuity of the IEL in developing collaterals. Collateral artery development is associated with permanent redistribution of existing elastic fibers to accommodate diameter growth. We found no evidence of new elastic fiber formation. Stabilization of the arterial wall during outward remodeling is necessary and dependent on LOX activity.

Endovascular management of complete disruption of aortic anastomosis after pediatric multivisceral transplant.

Multivisceral transplantation is a life-saving treatment for many chronically ill patients with advanced abdominal pathologies. For such transplants, a complex arterial reconstruction is required, with numerous anastomoses on a composite donor graft and the native aorta. In these patients, anastomotic disruption or pseudoaneurysm formation, often in the setting of infection, are deadly complications. Open surgical repair is hazardous, because many of these patients have dense adhesions. Reported cases of disruption at the aortic anastomosis to date have resulted in patient demise. We report the case of a pediatric multivisceral transplant recipient with ruptured aortic pseudoaneurysm. He underwent an emergent endovascular parallel stent grafting technique, which successfully controlled bleeding and maintained graft perfusion.

Persistent sciatic artery aneurysm treated with concomitant tibial bypass and vascular plug embolization.

The presence of a persistent sciatic artery is a rare congenital vascular malformation. Complications associated with aneurysmal degeneration of this aberrant vessel include rupture, thrombosis, and embolization with obliteration of outflow vessels. We describe the case of an 82-year-old female presenting with critical limb ischemia due to embolization from a partially thrombosed persistent sciatic artery aneurysm. Successful treatment was achieved via a common femoral to posterior tibial artery bypass with the great saphenous vein and vascular plug embolization of the aneurysm.

A modified rat model of hindlimb ischemia for augmentation and functional measurement of arteriogenesis.

Arteriogenesis (collateral artery development) is an adaptive pathway critical for salvage of tissue in the setting of arterial occlusion. Rodent models of arteriogenesis typically involve an experimental occlusion (ligation) of a hindlimb artery and then rely on indirect measures such as laser Doppler perfusion imaging to assess blood flow recovery. Unfortunately, the more commonly utilized measures of distal tissue perfusion at rest are unable to account for hemodynamic and vasoactive variables and thus provide an incomplete assessment of collateral network capacity. We provide a detailed description of modifications to the commonly used model of femoral artery ligation. These serve to alter and then directly assess collateral network's hemodynamic capacity. By incorporating an arteriovenous fistula distal to the arterial ligation, arterial growth is maximized. Hindlimb perfusion may be isolated to measure minimum resistance of flow around the arterial occlusion, which provides a direct measure of collateral network capacity. Our results reinforce that arteriogenesis is driven by hemodynamic variables, and it can be reliably augmented and measured in absolute terms. Using these modifications to a widely used model, functional arteriogenesis may be more directly studied.

A retrievable rescue stent graft and radiofrequency positioning for rapid control of noncompressible hemorrhage.

BACKGROUND: Noncompressible hemorrhage of the torso remains a challenging surgical dilemma. Stent graft repair requires endovascular expertise, imaging, and inventory that are not available within the critical window of massive hemorrhage. We developed a retrievable stent graft for rapid hemorrhage. We further investigated a radiofrequency (RF) positioning approach as a possible alternative to the logistics of fluoroscopy. METHODS: A retrievable stent graft was constructed with a novel "petal and stem" design from nitinol and covered with a sleeve of electrospun polyurethane. The stent graft was tested using an in vitro model of simulated hemorrhage. Next, the stent graft was examined in vivo using a porcine model of noncompressible hemorrhage. The stent was examined for hemorrhage control in a porcine model of either aortic or caval injury. An RF reader was assembled from an Arduino processor while RF tags were affixed to the ends of the stent graft. Detection accuracy of a handheld RF wand for an RF tag was quantified both in vitro and through tissue. RESULTS: The retrievable RESCUEstent graft was deployed within minutes and rapidly controlled traumatic hemorrhage angiographically in both aortic injury (n = 3) and caval injury (n = 2). Stent grafts were easily recaptured in both models in under 15 seconds. The LED light of a handheld RF detector illuminated when positioned directly over an RF tag. The RF detection approach revealed positioning accuracy to within 1 cm of the intended target, despite tissue interference. CONCLUSION: This study demonstrates the rapid deployment and retrieval of a RESCUE stent graft as well as the ability to tamponade injuries of the aorta and cava. In addition, this study demonstrates the feasibility of RF tags to guide stent placement through tissue. More rigorous models are needed to define the effectiveness of this approach in the setting of vascular injury and shock.