Ulka Sachdev

High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury

OBJECTIVES The angiogenic drive in skeletal muscle ischemia remains poorly understood. Innate inflammatory pathways are activated during tissue injury and repair, suggesting that this highly conserved pathway may be involved in ischemia-induced angiogenesis. We hypothesize that one of the endogenous ligands for innate immune signaling, high mobility group box 1 (HMGB1), in combination with autophagic responses to hypoxia or nutrient deprivation, plays an important role in angiogenesis. METHODS Human dermal microvascular endothelial cells (ECs) were cultured in normoxia or hypoxia (1% oxygen). Immunocytochemical analysis of HMGB1 subcellular localization, evaluation of tube formation, and Western blot analysis of myotubule light-chain 3I (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-Methyladenine (3MA), chloroquine, or rapamycin were administered to inhibit or promote autophagy, respectively. In vivo, a murine hind limb ischemia model was performed. Muscle samples were collected at 4 hours to evaluate for nuclear HMGB1 and at 14 days to examine endothelial density. Perfusion recovery in the hind limbs was calculated by laser Doppler perfusion imaging (LDPI). RESULTS Hypoxic ECs exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity, 186.9 ± 17.1 vs 236.0 ± 1.6, P = .01) with a concomitant increase in cytosolic staining. HMGB1 treatment of ECs enhanced tube formation, an angiogenic phenotype of ECs. Neutralization of endogenous HMGB1 markedly impaired tube formation and inhibited LC3II formation. Inhibition of autophagy with 3MA or chloroquine abrogated tube formation, whereas its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced numbers of HMGB1-positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9% vs 51.7% ± 2.0%, P < .001). Injection of HMGB1 into ischemic hind limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1 vs 34.2 ± 3.4 ECs/high-powered field, respectively; P = .02) at 14 days compared with control hind limbs. CONCLUSIONS Nuclear release of HMGB1 and autophagy occur in ECs in response to hypoxia or serum depletion. HMGB1 and autophagy are necessary and likely play an interdependent role in promoting the angiogenic behavior of ECs. In vivo, HMGB1 promotes perfusion recovery and increased EC density after ischemic injury. These findings suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis.

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling. Methods Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism. Results While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro. Discussion TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

Toll-like receptors (TLRs) play an important role in regulating muscle regeneration and angiogenesis in response to ischemia. TLR2 knockout mice exhibit pronounced skeletal muscle necrosis and abnormal vessel architecture after femoral artery ligation, suggesting that TLR2 signaling is protective during ischemia. TLR4, an important receptor in inflammatory signaling, has been shown to regulate TLR2 expression in other systems. We hypothesize that a similar relationship between TLR4 and TLR2 may exist in hindlimb ischemia in which TLR4 upregulates TLR2, a mediator of angiogenesis and perfusion recovery. We examined the expression of TLR2 in unstimulated and in TLR-agonist treated endothelial cells (ECs). TLR2 expression (low in control ECs) was upregulated by lipopolysaccharide, the danger signal high mobility group box-1, and hypoxia in a TLR4-dependent manner. Endothelial tube formation on Matrigel as well as EC permeability was assessed as in vitro measures of angiogenesis. Time-lapse imaging demonstrated that ECs lacking TLR4 formed more tubes, whereas TLR2 knockdown ECs exhibited attenuated tube formation. TLR2 also mediated EC permeability, an initial step during angiogenesis, in response to high-mobility group box-1 (HMGB1) that is released by cells during hypoxic injury. In vivo, ischemia-induced upregulation of TLR2 required intact TLR4 signaling that mediated systemic inflammation, as measured by local and systemic IL-6 levels. Similar to our in vitro findings, vascular density and limb perfusion were both enhanced in the absence of TLR4 signaling, but not if TLR2 was deleted. These findings indicate that TLR2, in the absence of TLR4, improves angiogenesis and perfusion recovery in response to ischemia.

Autologous alternative veins may not provide better outcomes than prosthetic conduits for below-knee bypass when great saphenous vein is unavailable

BACKGROUND There is a need to better define the role of alternative autologous vein (AAV) segments over contemporary prosthetic conduits in patients with critical limb ischemia when great saphenous vein (GSV) is not available for use as the bypass conduit. METHODS Consecutive patients who underwent bypass to infrageniculate targets between 2007 and 2011 were categorized in three groups: GSV, AAV, and prosthetic. The primary outcome was graft patency. The secondary outcome was limb salvage. Cox proportional hazards regression was used to adjust for baseline confounding variables. RESULTS A total of 407 infrainguinal bypasses to below-knee targets were analyzed; 255 patients (63%) received a single-segment GSV, 106 patients (26%) received an AAV, and 46 patients (11%) received a prosthetic conduit. Baseline characteristics were similar among groups, with the exception of popliteal targets and anticoagulation use being more frequent in the prosthetic group. Primary patency at 2 and 5 years was estimated at 47% and 32%, respectively, for the GSV group; 24% and 23% for the AAV group; and 43% and 38% for the prosthetic group. Primary assisted patency at 2 and 5 years was estimated at 71% and 55%, respectively, for the GSV group; 53% and 51% for the AAV group; and 45% and 40% for the prosthetic group. Secondary patency at 2 and 5 years was estimated at 75% and 60%, respectively, for the GSV group; 57% and 55% for the AAV group; and 46% and 41% for the prosthetic group. In Cox analysis, primary patency (hazard ratio [HR], 0.55; P < .001; 95% confidence interval [CI], 0.404-0.758), primary assisted patency (HR, 0.57; P = .004; 95% CI, 0.388-0.831), and secondary patency (HR, 0.56; P = .005; 95% CI, 0.372-0.840) were predicted by GSV compared with AAV, but there was no difference between AAV and prosthetic grafts except for the primary patency, for which prosthetic was protective (HR, 0.38; P < .001; 95% CI, 0.224-0.629). Limb salvage was similar among groups. CONCLUSIONS AAV conduits may not offer a significant patency advantage in midterm follow-up over prosthetic bypasses.

MyD88 and TRIF mediate divergent inflammatory and regenerative responses to skeletal muscle ischemia.

We have previously shown that MyD88 KO mice appear protected from ischemic muscle injury while TRIF KO mice exhibit sustained necrosis after femoral artery ligation (FAL). However, our previous data did not differentiate whether the protective effect of absent MyD88 signaling was secondary to attenuated injury after FAL or quicker recovery from the insult. The purpose of this study was to delineate these different possibilities. On the basis of previous findings, we hypothesized that MyD88 signaling promotes enhanced inflammation while TRIF mediates regeneration after skeletal muscle ischemia. Our results show that after FAL, both MyD88 KO mice and TRIF KO mice have evidence of ischemia, as do their control counterparts. However, MyD88 KO mice had lower levels of serum IL‐6 24 h after FAL, while TRIF KO mice demonstrated sustained serum IL‐6 up to 1 week after injury. Additionally, MyD88 KO mice had higher nuclear content and larger myofibers than control animals 1 week after injury. IL‐6 is known to have differential effects in myoblast function, and can inhibit proliferation and differentiation. In tibialis anterior muscle harvested from injured animals, IL‐6 levels were higher and the proliferative marker MyoD was lower in TRIF KO mice by PCR. Furthermore, expression of MyD88 appeared to be higher in skeletal muscle of TRIF KO mice. In vitro, we showed that myoblast differentiation and proliferation were attenuated in response to IL‐6 treatment giving credence to the finding that low IL‐6 in MyD88 KO mice may be responsible for larger myocyte sizes 1 week after FAL. We conclude that MyD88 and TRIF work in concert to mediate a balanced response to ischemic injury.

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.

Perpetual change: autophagy, the endothelium, and response to vascular injury

Current studies of vascular health, aging, and autophagy emphasize how the endothelium adapts to stress and contributes to disease. The endothelium is far from an inert barrier to blood‐borne cells, pathogens, and chemical signals; rather, it actively translates circulating mediators into tissue responses, changing rapidly in response to physiologic stressors. Macroautophagy—the cellular ingestion of effete organelles and protein aggregates to provide anabolic substrates to fuel bioenergetics in times of stress—plays an important role in endothelial cell homeostasis, vascular remodeling, and disease. These roles include regulating vascular tone, sustaining or limiting cell survival, and contributing to the development of atherosclerosis secondary to infection, inflammation, and angiogenesis. Autophagy modulates these critical functions of the endothelium in a dynamic and perpetual response to tissue and intravascular cues.

Cephalic vein transposition is a durable approach to managing cephalic arch stenosis

INTRODUCTION The proximal cephalic vein that enters the axillary vein (cephalic arch) is a common site of stenosis in patients with upper extremity arteriovenous fistulas for hemodialysis (HD). In this study, we present the outcomes of a series of cephalic vein transposition, to determine its utility in the setting of refractory arch stenosis. METHODS We conducted a retrospective review of patients undergoing cephalic vein transposition to manage refractory cephalic arch stenosis from January 1, 2008 to August 31, 2015. Demographics, past medical history, access history of the patients as well as procedural details of the surgery to the stenotic segment, patency of the access, and the need for subsequent interventions were recorded. RESULTS Twenty-three patients underwent a cephalic vein transposition during the study period. The patients undergoing cephalic transposition had their current access for an average of 3.0 ± 2.6 years and had an average of 2.3 ± 0.9 interventions on the access prior to the surgery. Complications from the surgery were uncommon (8.7%) and no patient required a temporary tunneled dialysis catheter. The re-intervention rate was 0.2 ± 0.2 interventions per patient per year. At two years, primary patency was 70.9% and cumulative patency was 94.7% for the patients with cephalic transposition. CONCLUSIONS Cephalic vein transposition is safe and effective treatment for cephalic arch stenosis without interrupting utilization of the access. The surgical approach to stenosis of the proximal cephalic vein is effective, requires minimal re-interventions, and should be considered for isolated, refractory cephalic arch stenosis in mature arteriovenous fistulas.

Suppressed networks of inflammatory mediators characterize chronic venous insufficiency

OBJECTIVE Chronic venous insufficiency (CVI) affects 25 million adults in the United States. Little emphasis has been placed on inflammatory changes associated with CVI. We hypothesize that in patients with early to mid-stage benign varicose vein disease, differences in circulating inflammatory mediators will be manifested in blood draining the involved area vs circulating blood in control subjects. METHODS Patients undergoing either endovenous ablation or sclerotherapy for Clinical, Etiology, Anatomy, and Pathophysiology clinical class 3 to 5 disease underwent phlebotomy from regional veins at the time of the procedure. The patients age, gender, clinical class, duration of symptoms, presence of superficial truncal reflux by duplex ultrasound, and treatment modality were recorded. Plasma from patients and banked blood samples from healthy volunteers (HVs) were subjected to Luminex (EMD Millipore, Billerica, Mass) to evaluate the expression of an established panel of 20 inflammatory mediators. Mediator concentrations were compared between patients and HVs using Mann-Whitney U tests. Importantly, computational analysis allowed us to compare not only the panel of inflammatory mediators but also the inflammatory networks connecting these mediators to one another. Principal components were analyzed to assess network robustness in each group. RESULTS CVI venous blood revealed significantly lower levels of monokine induced by γ interferon, soluble interleukin (IL) 2 receptor α chain, IL-4, IL-6, IL-7, tumor necrosis factor α, eotaxin, and granulocyte-macrophage colony-stimulating factor than blood from controls. Inflammatory networks were significantly less complex and less robust in the CVI patients compared with HVs. Based on principal component analysis, responses among HVs were more varied than those of CVI patients. CONCLUSIONS We demonstrate that patients with CVI have significant differences not only in blood-borne inflammatory mediators but also in the interconnectedness of these mediators with one another and in their principal inflammatory characteristics. Results suggest hypoinflammation in chronic nonhealing changes in CVI. These novel findings, if validated in larger cohorts, may help predict the risk of disease progression or response to therapy in the future and may guide mechanistic studies on tissue responses to CVI.

Extended Treatment with Chloroquine, an Inhibitor of Autophagy, Disrupts Angiogenic Drive in Muscle after Arterial Ligation Without Detrimental Effects on Muscle Inflammation and Regeneration

INTRODUCTION: We showed that in ischemicmuscle, short-term use of chloroquine (CQ), a common drug that inhibits autophagosome fusion with lysosomes, decreases early markers of autophagy, a stressinduced protective mechanism to recycle organelles. We hypothesized that prolonged chloroquine treatment will produce marked inflammation,poor regeneration, anddisrupted angiogenesis aftermuscle ischemia.