Femoral Ligation Research Articles

Changes in the demographics of intravenous drug users with mycotic common femoral artery pseudoaneurysm as a consequence of self-injection does not influence outcome following emergency ligation.

Objectives The reported annual incidence of mycotic pseudoaneurysm of the common femoral artery in intravenous drug users has been estimated at 0.03%. Over the past 5 years in Scotland, the proportion of people receiving specialist attention for heroin use over the age of 40 years has increased from 15 to 22%. Although routinely managed with arterial ligation (without reconstruction), some series have reported rates of major limb amputation of up to 10%. We sought to define whether this management strategy was still acceptable in an older population. Methods Retrospective review of patients presenting to a tertiary vascular service with mycotic pseudoaneurysm of the common femoral artery due to arterial injection by intravenous drug users between October 2010 and March 2016. Variables of interest included patient demographics and requirement for major amputation. Results There were 55 patients identified. The annual incidence of mycotic pseudoaneurysm of the common femoral artery in intravenous drug users was 2.1%. It was more common in men (3:1) and the mean age at presentation was 41 years (standard deviation ± 8 years). Three patients underwent major limb amputation during the index admission for severe limb ischaemia (two transfemoral amputations; one hip-disarticulation). Following discharge two patients were readmitted (134 and 200 days, respectively, following primary ligation) for major limb amputation due to of critical limb ischaemia. Conclusions Despite the increasing age of intravenous drug users presenting with mycotic pseudoaneurysm of the common femoral artery primary ligation of pseudoaneurysm would seem to remain an appropriate therapeutic intervention.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Surgical outcome after femoral vessels ligation in I/V drug users

Intravascular drug abuse is being practiced with increasing frequency in our country, resulting in dreadful complications. A common complication of intravenous drug users is traumatic damage to tissues and organs secondary to injections, which depends on a number of factors which are difficult to quantify; local drug practices, the skill of the injector and the susceptibility of the individual. Objective: To study the incidence, limb complications and indications for limb amputation in intravenous drug abusers. Methods: A descriptive study on nineteen intravenous drug abusers was carried out at department of Surgery, Mayo hospital Lahore. Results: Entire limb survived in Eleven patients, Four had toe amputations, Three ended in below knee amputation and One in above knee amputation. Conclusion: Most of the affected limbs can survive despite the major vessels being tied due to development of collateral circulation.

The Secretome of Hydrogel-Coembedded Endothelial Progenitor Cells and Mesenchymal Stem Cells Instructs Macrophage Polarization in Endotoxemia.

: We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid-based (HA)-hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC-MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA-hydrogel-embedded delivery of cells to LPS-induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC-MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned medium changed their polarization to anti-inflammatory M2. Incubation of coembedded EPCs-MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti-inflammatory interleukin (IL)-4 and IL-10. EPC-MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs-MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned. This report provides insight into a new therapeutic approach for treatment of sepsis and provides a new and improved strategy using hydrogels for the delivery of stem cells to treat sepsis and, potentially, other injuries and/or diseases. The delivery of two different stem cell lines (endothelial progenitor cells and mesenchymal stem cells; delivered alone and together) embedded in a protective bioengineered scaffolding (hydrogel) offers many therapeutic benefits for the treatment of sepsis. This study shows how hydrogel-delivered stem cells elicit their effects and how hydrogel embedding enhances the therapeutic efficacy of delivered stem cells. Hydrogel-delivered stem cells influence the components of the overactive immune system during sepsis and work to counterbalance the release of many proinflammatory and prodamage substances from immune cells, thereby improving the associated vascular and kidney damage.

Femoral artery occlusion amplifies TRPA1 function in skeletal muscle sensory neurons (1132.5)

A prior study showed that transient receptor potential channel A1 (TRPA1) in thin‐fiber muscle afferents contributes to the amplified reflex autonomic responses observed in rats with femoral artery ligation. Thus, in this study we characterized current responses induced by activation of TRPA1 in skeletal muscle dorsal root ganglion (DRG) neurons of control limbs and limbs with 24 hrs of femoral ligation using whole‐cell patch clamp methods. Our results show that AITC (a TRPA1 agonist, 50‐200 µM) produced a dose‐dependent increase of amplitudes of inward current responses. Also, the peak current amplitude induced by AITC is significantly larger in DRG neuron of ligated limbs. There is no difference in size distribution of DRG neurons with AITC‐induced current responses between two groups. However, arterial occlusion increases the percentage of the AITC‐sensitive DRG neurons compared with control. AITC‐induced currents in DRG neurons are significantly attenuated by exposure to 10 µM of HC‐030031, a selective inhibitor of TRPA1. In addition, capsaicin (a TRPV1 agonist) evokes a greater increase in the amplitude of AITC‐currents in DRG neurons of ligated limbs. Overall, our data suggest that a greater TRPA1 function is developed in muscle afferent nerves with the hindlimb ischemia and TRPV1 is partly responsible for augmented TRPA1 responses induced by arterial occlusion.Grant Funding Source: NIH P01 HL096570 and AHA EIA 0840130N

Abstract 52: Statin Therapy Accelerates Venous Thrombus Resolution: Assessment In Stasis And Chemical Injury Induced Thrombosis

Introduction In deep vein thrombosis (DVT), both the induced inflammation response and fibrinolytic capacity are critical in resolving experimental DVT. Unresolved, obstructing thrombi can lead to the burdensome and costly sequelae of the post-thrombotic syndrome. Recently, statins have shown to reduce the incidence of DVT (JUPITER trial). In this study, we investigated the in vivo effects of statin therapy on resolution of already formed DVT using serial intravital microscopy (IVM) imaging and thrombus mass measurements. Methods DVT was induced in C57Bl/6J male mice (N=190) by topical ferric chloride of the femoral vein (chemical injury) or ligation of the IVC (stasis). One day after DVT induction, mice started treatment of atorvastatin (1.14mg/kg), mevastatin (10 mg/kg) or PBS by once daily oral gavage. Serial IVM was preformed at days 2, 4 and 6 utilizing FITC-dextran (ex/em 490/520nm), CLIO-AF555 (ex/em 555/565nm), and MMPSense680 (ex/em 680/700nm) imaging agents for thrombus architecture, macrophage content and MMP activity measurements. Thrombus masses were collected at days 4, 7, 10 in stasis DVT mice and combination statin/enoxaparin (10mg/kg/d, s.c.) were also compared to statin therapy. Platelet activation and coagulation were analyzed using aggregometry and thromboelastography on whole-blood. Results We found that statin therapy reduced DVT burden and inflammation in both chemical- and stasis-induced established DVT up to 10 days compared to control (p<0.05). Serial IVM imaging of statin animals showed an increase in DVT resolution from day 2 to 4 (Δlength -21± 6.0 vs. control -7.1± 5.6%, p<0.01), and decreased inflammation compared to controls (p<0.001). Inflammation was also found to predict the extent of DVT resolution(r=0.84). Statin therapy also reduced thrombus plasminogen activator inhibitor-1, tissue factor, and Mac-3 levels, as well as reducing platelet activation and clot strength(p<0.05). Conclusion Statin therapy accelerates DVT resolution despite reducing thrombus related inflammation. The net reduction of thrombus burden tracked with statin-mediated favorable effects on fibrinolysis, coagulation, and platelet function. These results support statins as a translatable therapy to improve the resolution of DVT.

Needle Embolisation to the Right Ventricle: Multiple Complications in a Complex Patient

IntroductionThe communal and repeated use of injection needles predisposes injecting drug users to several complications. ReportThis case report describes uncommon cardiac and pulmonary complications in a 44-year old male, injecting drug abuser. The retention of fractured injection needles in the groin, and the subsequent embolisation to the right ventricle, predisposed to recurrent systemic infections, and cavitating pulmonary septic emboli and pleural empyema as well as local complications. Years later, the needle was completely embedded in the wall of the right ventricle and not suitable for transvenous removal. DiscussionContinuing intravenous drug abuse with injections into the groin result in infective complications, commonly pseudoaneurysms of the distal common femoral artery requiring triple femoral ligation. The embolisation of a fractured injection needle to the RV is a clinical rarity.

Toward a mouse model of hind limb ischemia to test therapeutic angiogenesis

Several clinical trials are currently evaluating stem cell therapy for patients with critical limb ischemia that have no other surgical or endovascular options for revascularization. However, these trials are conducted with different protocols, including use of different stem cell populations and different injection protocols, providing little means to compare trials and guide therapy. Accordingly, we developed a murine model of severe ischemia to allow methodic testing of relevant clinical parameters. High femoral artery ligation and total excision of the superficial femoral artery was performed on C57BL/6 mice. Mononuclear cells (MNCs) were isolated from the bone marrow of donor mice, characterized using fluorescence-activated cell sorting, and injected (5×10(5) to 2×10(6)) into the semimembranosus (proximal) or gastrocnemius (distal) muscle. Vascular and functional outcomes were measured using invasive Doppler imaging, laser Doppler perfusion imaging, and the Tarlov and ischemia scores. Histologic analysis included quantification of muscle fiber area and number as well as capillary density. Blood flow and functional outcomes were improved in MNC-treated mice compared with controls over 28 days (flow: P<.0001; Tarlov: P=.0004; ischemia score: P=.0002). MNC-treated mice also showed greater gastrocnemius fiber area (P=.0053) and increased capillary density (P=.0004). Dose-response analysis showed increased angiogenesis and gastrocnemius fiber area but no changes in macroscopic vascular flow or functional scores. Overall functional outcomes in mice injected proximally to the ischemic area were similar to mice injected more distally, but muscle flow, capillary density, and gastrocnemius fiber area were increased (P<.05). High femoral ligation with complete excision of the superficial femoral artery is a reliable model of severe hind limb ischemia in C57BL/6 mice that shows a response to MNC treatment for functional and vascular outcomes. A dose response to the injection of MNCs appears to be present, at least microscopically, suggesting that an optimal cell number for stem cell therapy exists and that preclinical testing needs to be performed to optimally guide human trials. Injection of MNCs proximal to the site of ischemia may provide different outcomes compared with distal injection and warrants additional study.

Application of colour Doppler in encircling constriction of the superficial femoral vein in primary deep venous insufficiency of the lower limbs

1. The aim of the present study was to evaluate the clinical value of colour Doppler application in encircling constriction of the superficial femoral vein in deep vein insufficiency. 2. A total of 87 patients with primary deep venous insufficiency (PDVI) using ascending venography were randomly divided into group A (44 patients) and group B (43 patients). All patients underwent encircling constriction of the superficial femoral vein, high ligation and ablation of the great saphenous vein and perforator vein. The duration of venous reflux at operation was monitored with colour Doppler in group A (but not group B) to evaluate the immediate effects. Clinical grading and scoring of the clinical, etiological, anatomical, pathophysiological (CEAP) classification system were used to evaluate the follow-up curative effect. 3. In four cases from group A, completely destroyed valves were identified at the time of operation and autografting of the vein segment with a valve was carried out. The intraoperative examination of colour Doppler in group A showed a much shorter duration of vein reflux after the encircling constriction procedure than the presurgery condition. According to the results of CEAP grading, the success rate of group A (95.0%, 38/40) was significantly higher than that of group B (76.7%, 33/43). Postoperative clinical scores were markedly lower than preoperative scores in both groups A and B. 4. In conclusion, our data suggest that application of colour Doppler in encircling constriction of superficial femoral vein might enhance surgical pertinence and improve surgical effect for PDVI.

Model of osteonecrosis of the femoral head created by percutaneous transluminal medial femoral circumflex arterial ligation

研究发现,髋关节牵引可导致旋股内、外动脉尤其股骨头供养血管闭塞与血运障碍[1-3],为了观察持续牵引、闭塞这些血管对股骨头血循环的影响尤其会否发生股骨头坏死(osteonecrosis of the femoral head,ONFH),笔者进行了经皮经腔犬旋股内动脉内结扎术(percutaneous transluminal medial femoral circumfflex arterial ligation,PTMFCAL)的研究,结果报告如下。

Contribution of nerve growth factor to upregulation of P2X3 expression in DRG neurons of rats with femoral artery occlusion

Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X(3) receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X(3) in dorsal root ganglion (DRG) neurons of rats. Also, P2X(3)-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X(3) was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X(3) (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X(3) expression in DRG neurons (percentage of P2X(3)-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,β-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X(3) (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,β-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,β-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X(3) in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X(3) receptor expression and response.

Nerve growth factor contributes augmented P2X3 expression of primary afferent neurons by femoral occlusion

Femoral artery occlusion augments the reflex sympathetic nerve and pressor responses to muscle metabolites and to muscle contraction in rats. Also, P2X3 receptors contribute the metabolic component of the exercise pressor reflex. Thus, the purpose of this study was to examine if femoral occlusion alters expression of P2X3 in dorsal root ganglion (DRG) neurons and its responsiveness in rats. In addition, we examined role of nerve growth factor (NGF) in the levels of P2X3 proteins in sensory neurons. Our western blot results showed that 24hrs of femoral ligation increased the levels of P2X3 (optical density: 0.93±0.07 in control vs. 1.37±0.10 after occlusion, P<0.05). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X3 expression in DRG neurons (percentage of P2X3 positive cells: 33±3% in control vs.51±3% in occlusion, P<0.05). Furthermore, our results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than in control rats by injection of α,β me-ATP into the arterial blood supply of hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X3 (optical density: 0.98±0.12 in control vs. 1.37±0.16 with NGF, P<0.05). Our findings suggest that the levels of P2X3 in primary afferent neurons are increased as the blood supply to the hindlimb is insufficient, and NGF is likely responsible for enhanced P2X3. (Supported by NIH R01 HL-090720, P01HL-096570, and AHA grant 0840130N).

Human UCB CD133+ Cell Infusion In NOD-SCID Mice with Acute Hind Limb Ischemia Improves Reperfusion of An Ischemic Bed

AbstractAbstract 4697Clinical trials have shown intracoronary or intramuscular injections of hematopoietic stem cells (HSC) ameliorate vascular ischemia. HSCs are isolated from bone marrow (BM), umbilical cord blood (UCB), and adult peripheral blood (AB). Critical limb ischemia patients, injected with autologous HSC into ischemic tissue had improved perfusion and reduced amputations. However, autologous HSC effectiveness declines with age. We hypothesized that UCB CD133+ HSCs' are more efficacious than autologous HSC at revascularization for an ischemic bed.Immune reactive surface antigens on UCB CD133+ cells were determined using multi-parameter flow cytometry. UCB CD133+ allogeneic immune responses from 72 h co-cultures were analyzed using a mixed lymphocyte culture (MLR) proliferation assays, and induced cytokine production found using TH1(inflammatory)/TH2(anti-inflammatory) cytometric bead assays (CBA) (BD Biosciences (San Jose, CA). In vivo studies were done using NOD/SCID mice treated with low-dose irradiation (200cGy) then femoral ligation. Study mice were divided into 3 treatment groups and underwent intracoronary infusions of; 1×106 UCB CD133+, 0.5×106 UCB mononuclear cells (MNCs), or EGM media alone. Day 3 post-ligation and cell infusion, quadriceps from 3 mice in each group were harvested, total RNA isolated using Trizol™ and RNA for each treatment group pooled. Pooled RNA was converted to cDNA then assayed using mouse ABI Affymetrix™ Gene chip. Computational pathway analyzes were run for significant transcriptome changes. Gene array data was confirmed on select genes using quantitative reverse transcriptase real time PCR (qPCR). Treated mice were followed for ischemic bed reperfusion by Laser Doppler imaging for 28 days post-ligation. UCB CD133+ cells express MHC class I and II, but lack CD80 and CD86. Allogeneic MLR assays using UCB and AB MNCs showed UCB CD133+ cells were stimulatory to MNC proliferation. The MNCs alloresponse to UCB CD133+ cells produced high concentration of interleukin 4 (IL-4) and IL-10 indicative of a TH2 response. These results show allogeneic UCB CD133+ cells induce a TH2 immune response due to an absence of co-stimulation of immune cells. To verify whether the results are recapitulated in vivo, we treated acute limb ischemia in NOD/SCID mice with human UCB CD133+ cells and monitored revascularization by blood flow and Affymetrix mouse gene array. Computational analysis of Affymetrix mouse gene arrays from comparing all 3 treatment protocols indicated that ischemic quadriceps treated with UCB CD133+ cells exhibited a significant enrichment of anti-angiogeneic, chemotaxic, cytokine, and immune response genes. UCB CD133+ treated mice had increase expression of anti-inflammatory, and anti-angiogeneic genes versus controls. Conversely media and UCB MNC treated mice had equal to or higher pro-inflammatory gene expression. The anti-inflammatory gene response was confirmed for 2 of the 3 genes, arginase 1, CXCL7, and tumor growth factor beta binding protein 2 (TGFβBP2) comparing CD133+ treated mice to UCB MNC treated mice. TGFβBP2 was not observed to change in vivo. UCB CD133+ cells treated mice also had improved blood perfusion of the ischemic bed at 28 days post-ligation versus control protocols. The in vivo mouse model supports the in vitro observation, but demonstrates improved reperfusion over controls.Currently there are 2 non-exclusive hypotheses for the function of CD133+ cells in revascularization. First the CD133+ cells integrate and differentiate into new vascular endothelial cells and the second is that the CD133+ cells augment revascularization via a paracrine effect which does not require integration into the host tissue. In vitro studies suggest that UCB CD133+ cells promote an allogeneic TH2 type response because of a lack of co-stimulatory immune surface antigens, resulting in high concentrations of IL-4 and IL-10. This skewed TH2 response inhibits inflammation, the subsequent tissue damage, and may augment revascularization. Although these studies cannot exclude a role for UCB CD133+ cellular integration, results support the paracrine hypothesis as infusion of selected CD133+ cells appears to promote an anti-inflammatory, anti-angiogenic profile at day 3 post-ligation and at 1 month improved reperfusion compared with non-selected MNC. Disclosures:No relevant conflicts of interest to declare.

Hypoxia Preconditioned Mesenchymal Stem Cells Improve Vascular and Skeletal Muscle Fiber Regeneration After Ischemia Through a Wnt4-dependent Pathway

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.

Strain-dependent variation in collateral circulatory function in mouse hindlimb

The extent (density and diameter) of the native (preexisting) collateral circulation in healthy tissues and the capacity of collaterals to enlarge/remodel in obstructive arterial disease are important determinants of ischemic injury. Evidence suggests that these parameters vary widely from yet-to-be-identified genetic and environmental factors. Recently, a locus on chromosome 7 was linked to less recovery of perfusion after femoral artery ligation in BALB/c and A/J versus C57BL/6 mouse strains. Moreover, evidence suggested that BALB/c and A/J share an allele(s) at this locus that is different from C57BL/6 mice. Here we tested the hypothesis that differences in collateral extent and/or remodeling underlie these findings. Compared with C57BL/6, BALB/c and A/J strains have fewer native collaterals in hindlimb (also confirmed in brain)-associated with greater reduction in perfusion immediately after femoral ligation, slower recovery of perfusion, greater hindlimb use impairment, and worse ischemia. However, A/J also differed from BALB/c in a number of these parameters, including having more robust collateral remodeling. Analysis of A/J --> C57BL/6 chromosome substitution strains confirmed that a difference in an allele(s) on chromosome 7 conferred most, but not all, of the magnitude of the differences in collateral function. Additional studies of C57BL/6 x BALB/c F1 mice demonstrated that alleles of the C57BL/6 strain exert dominance for collateral traits. Finally, negative results were obtained from studies examining a previously identified candidate gene potentially responsible for these differences-Bcl2-associated athanogene-3. These findings emphasize the major contribution of genetic background to variation in the collateral circulation and its capacity to lessen ischemia in obstructive disease.

Suppressed hindlimb perfusion in Rac2-/- and Nox2-/- mice does not result from impaired collateral growth.

While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.

Augmentation of Murine Vasculogenesis by Injected Human Hematopoietic Stem Cells in Response to Ischemia Follows Early Activation of Innate Immune Response

Although NOD.SCID mice lack an adaptive immune response, their innate immune system is intact. NOD.SCID mice are the model system used for both allo- and xeno-graft cell therapies to in vivo neovascularization of ischemic beds. As most previous studies have used undefined or mixed cell population, we tested the hypothesis that selected human umbilical cord blood (UCB) CD133+ stem cells (human HSC) would promote in vivo neovascularization, thereby conferring a therapeutic advantage over the mixed cell population found in mononuclear cell (MNC) preparations. These experiments focused on the murine response within the ischemic bed to human HSC cells as evaluated by gene array analyses. Gene array data was mined for inflammation, innate immunity and wound repair genes, with a focus on factors favorable to regeneration, angiogenesis and limits collagen deposition (scar formation). Normal mammalian wound repair occurs in 3 overlapping stages inflammation, new tissue formation, and remodeling. To evaluate these cell populations, NOD.SCID mice underwent femoral ligation and were divided into 3 study groups: controls (media alone), human selected UCB CD133+ HSC, or human non-selected UCB MNC. Previously, laser Doppler studies after femoral ligation showed significantly improved blood flow ratio in animals treated with human HSC vs. controls, 0.55±0.06 (n=9) and 0.37±0.03 (n=12, p<0.05), respectfully, and histology confirmed increased capillary density in animals treated with human HSC compared to controls, (320±18 and 131±6.9 cells/mm2; respectively p<0.0001). To compare the treatment effects on the early transcriptome and to wound repair, murine gene expression microarrays (Affymetrix murine GeneChip 430 2.0) of RNA extracted from tissues harvested from the ischemic limbs 3 days following femoral ligation and injection of cells (n=3 each experimental group) were utilized. In comparing control mice to those receiving human selected CD133+ HSC, 414 genes with 2-fold or greater change was observed. Several genes were differentially regulated between study groups. The level of IL12a mRNA was 2-fold lower in the hindlimb tissue of mice treated with CD133+ HSC than in the untreated controls: IL12 is a known regulator of the TH1-associated cytokine INF-g and TNF-a, a primary regulator of immune cells and cytolytic stimuli. While there was no observed change in the levels of INF-g in the hindlimb, 17 interferon induced genes were observed to be significantly lower in the human HSC-treated hindlimb compared to controls. Genes induced by TGF-b were lower including IL-12a, Ras-related genes, and the inhibitor latent transforming growth factor beta binding protein 2 was 3-fold higher in the hindlimbs treated with CD133+ HSC cells compared to controls. Innate immune cytokine receptors IL4 and IL13 mRNAs were expressed at a lower level than that seen in control mice. However, the IL6 receptor increased 3-fold in the human selected CD133+ HSC-treated cells as well as the downstream genes TIMP-1 (tissue inhibitor of matrix metalloprotease) and Pim1 gene, a stressed induced serine-threonine kinase which promotes cell survival. These results confirm prior observations in IL-6−/− knockout mice demonstrating delayed responses in leukocyte infiltration, angiogenesis and tissue remodeling. In addition, there was significant increases in genes regulated by the Wnt signaling pathway, compared to hindlimbs treated with UCB MNC cells including: Jun (2-fold higher), Sox4 (2–5 fold higher), Frizzle homolog 8 (2-fold), and MAPKKK4 (2-fold higher). The modulation of these factors and the observation that the inhibitor of Wnt signaling dickkopf was reduced 2.3-fold may indicate enhanced regenerative mechanisms as Wnt functions during embryogenesis and also during adult limb formation during metamorphosis. In summary, NOD.SCID treated with human selected CD133+ HSC after femoral ligation demonstrate significantly improved capillary density and blood flow recovery at later time points (day 28–42). Receptors and cytokines regulating innate immunity are significantly higher at an early time point (day 3) in mice treated with human CD133+ HSC suggesting an important regulatory role for innate inflammatory responses contributing to augmentation of vasculogenesis after acute vascular injury.

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins α5β1 and vβ3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins α5β1 and αvβ3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both α5β1 and αvβ3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was α5β1 and αvβ3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), α5β1, and αvβ3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.

Abstract 3595: Chloride Intracellular Channel-4 Determines Native Collateral Density

The density of native (pre-existing) collaterals in healthy tissue and their capacity to enlarge in occlusive disease (arteriogenesis) are major determinants of tissue injury in ischemia. Mechanisms regulating arteriogenesis remain unclear. Moreover, nothing is known about what specifies formation of native collaterals, except our report that VEGF contributes to native collateral formation (Clayton et al FASEB J 21:LB352, 2007). Recently, chloride intracellular channel proteins (CLICs) have been implicated in cellular processes including cell hollowing. CLIC1 and CLIC4 are expressed by endothelial cells, and VEGF-induced cell hollowing is critical for vascular formation in the embryo and angiogenesis in ischemia. Therefore, we examined CLIC1−/− and CLIC4−/− (null) mice for effects on native collateral density in healthy tissues and on arteriogenesis and angiogenesis in a hindlimb model of ischemia. Responses of CLIC1−/−mice did not differ from wild-type in any experiment. In contrast, CLIC4−/− showed a greater reduction in perfusion immediately after femoral ligation (p&lt;0.05 here and all statements below). Since baseline capillary density and collateral diameter were not different from wild-type, this suggested CLIC4−/− have reduced native collateral density. This was confirmed angiographically. In agreement, during 10 days after ligation CLIC4−/− mice had worse recovery of perfusion, greater ischemia and atrophy, and more impaired limb use, whereas collateral remodeling was unaffected. While ischemia increased expression of CLIC1 and CLIC4 similarly in wild-type mice, CLIC1 was augmented 3-fold in CLIC4−/−, suggesting CLIC1 may compensate for absence of CLIC4. Despite greater ischemia in CLIC4−/− mice, HIF1α and VEGF increased less in CLIC4−/−, suggesting CLIC4 may reside upstream of Hif1α-VEGF signaling. Native collateral density in the cerebral circulation of CLIC4−/− mice (but not CLIC1−/−) was also greatly reduced, resulting in 4-fold larger infarctions. This reduced collateral density in adults was associated with reduced formation and stabilization of nascent collaterals in the newborn. Hence, CLIC4 represents the second gene identified, besides VEGF, that specifies formation of the native collateral circulation.

Animal models of arteriogenesis

Arteriogenesis is the process of growth of collateral arteries after the stenosis of a feeding artery. The mechanism varies among species. In dogs, occlusion of a coronary artery leads to the growth of muscular vessels, whereas in pigs only capillary vessels grow. Ligation of rabbit and mouse femoral arteries induce collateral artery growth in non‐ischemic areas. The vascular response is restricted to collateral segments and collateral ending branches remain quiescent. Collateral neo‐formation is a rare event. Leukocyte recruitment and vascular proliferation dominate the process in both species, but only rabbit collateral arteries develop a neo‐intima layer. Two experimental models illustrate the crucial roles of leukocytes and shear stress in arteriogenesis: Infusion of the leukocyte chemoattractant protein MCP‐1, and surgical creation of an arterio‐venous shunt, both after femoral ligation result in an exacerbated development of collateral arteries. In a hamster model of Bland‐White‐Garland syndrome (origin of the left coronary artery from the pulmonary artery), collateral arteries connecting the right and left coronary arteries appear spontaneously, creating a left‐to‐right shunt that allows perfusion of the left heart. This model supports the idea that shear stress regulates not only the growth, but also de formation of new collateral arteries during the postnatal life.