Laser Doppler Perfusion Research Articles

Abstract 1142: Effect Of Chronic E-cigarette Nicotine Exposure On A Mouse Model Of Hind-limb Ischemia

Background: The rise of e-cigarette use in the United States has been rapid, yet its impact on peripheral artery disease (PAD) remains largely unexplored. This study investigates the relationship between e-cigarette use and PAD using a mouse ischemic hindlimb model. Method: Mice aged 6-8 weeks were divided into four groups. Three groups inhaled e-cigarette vapor continuously for 14, 20, or 28 days prior, and for 28 days after hindlimb ischemia (HLI) surgery, using an InExpose system (Figure 1). A control group was exposed to room air (RA) before and after HLI. Laser-Doppler perfusion imaging tracked hindlimb blood flow, and functional deficits were assessed using the modified ischemia scale. Figure 1: Methodology Results: Blood perfusion in the ischemic hindlimb of the e-cigarette groups (EC-14, EC-20, EC-28) was significantly lower than the non-vaping group at 21 days post-surgery. In the EC-14 group, perfusion improved after 10 days of vaping cessation (Figure 2). Additionally, there was a reduction in platelet and leukocyte counts in the e-cigarette groups compared to RA, with no difference in red blood cells. Necrosis and claw damage were observed in EC-20 and EC-28 groups but not in non-vaping mice. Figure 2: Representative images of perfusion recovery measured by LDPI. Conclusion: Chronic e-cigarette nicotine exposure impairs blood flow recovery and causes necrosis in mice with hind limb ischemia, potentially due to perturbed angiogenesis and arteriogenesis. This study highlights the detrimental effects of e-cigarette nicotine on vascular health.

Characteristics of Inflammatory and Normal Endothelial Exosomes on Endothelial Function and the Development of Hypertension.

Endothelial dysfunction is associated with the development of hypertension. We hypothesize that inflammatory and normal endothelial exosomes play their roles by mediating endothelial function, and they induce endothelial angiogenesis through different signaling pathways. Endothelial cell-derived exosomes were isolated from the human umbilical vein endothelial cells (HUVECs) treated with (TExo) or without (CExo) tumor necrosis factor (TNF)-α. We monitored dermal microcirculation profiles in spontaneously hypertensive rats (SHRs) and WKY rats using a laser Doppler imager and a laser Doppler perfusion and temperature monitor. Tube formation, levels of angiogenesis-related proteins in HUVEC-conditioned media, and reactive oxygen species (ROS) levels were assessed following TNF-α, CExo, or TExo treatments. Western blot analysis was conducted to examine signaling proteins associated with inflammation and ROS. The results showed increased blood perfusion and the mean amplitude of endothelial oscillator in SHRs following CExo administration. TNF-α, CExo, and TExo treatments promoted endothelial tube formation and elevated levels of angiogenic factors and ROS. TExo significantly increased phosphorylation levels of STAT3, p38, and level of NF-κB, while decreasing phosphorylation levels of JNK and Erk (P < 0.01 or P < 0.05). CExo significantly increased STAT3 phosphorylation and reduced JNK and Erk phosphorylation (all P < 0.01). In conclusion, TNF-α and TExo induce inflammatory and pathological angiogenesis via the NF-κB pathway, while CExo exhibits a physiologically pro-angiogenic effect on endothelial cells. Increased ROS, interplaying with inflammatory signals, contribute to exosome-mediated alterations of endothelial function, thereby playing a role in the development of hypertension.

Low‑intensity pulsed ultrasound accelerates diabetic wound healing by ADSC‑derived exosomes via promoting theuptake of exosomes and enhancing angiogenesis.

Diabetic wounds remain a great challenge for clinicians globally as a lack of effective radical treatment often results in poor prognosis. Exosomes derived from adipose‑derived stem cells (ADSC‑Exos) have been explored as an appealing nanodrug delivery system in the treatment of diabetic wounds. However, the short half‑life and low utilization efficiency of exosomes limit their therapeutic effects. Low‑intensity pulsed ultrasound (LIPUS) provides a non‑invasive mechanical stimulus to cells and exerts a number of biological effects such as cavitation and thermal effects. In the present study, whether LIPUS could enhance ADSC‑Exo‑mediated diabetic wound repair was investigated and its possible mechanism of action was explored. After isolation and characterization, ADSC‑Exos were injected into mice with diabetic wounds, then the mice were exposed to LIPUS irradiation. The control mice were subcutaneously injected with PBS. Wound healing assays, laser Doppler perfusion, Masson's staining and angiogenesis assays were used to assess treatment efficiency. Then, ADSC‑Exos were cocultured with human umbilical vein endothelial cells (HUVECs), and the proliferation, migration and tube formation of HUVECs were assessed. Moreover, the cellular uptake of ADSC‑Exos invitro and invivo was assessed to explore the synergistic mechanisms underlying the effects of LIPUS. The invivo results demonstrated that LIPUS increased the uptake of exosomes and prolonged the residence of exosomes in the wound area, thus enhancing angiogenesis and accelerating wound repair in diabetic mice. The invitro results further confirmed that LIPUS enhanced the uptake efficiency of ADSC‑Exos by 10.93‑fold and significantly increased the proliferation, migration and tubular formation of HUVECs. Therefore, the present study indicates that LIPUS is a promising strategy to improve the therapeutic effects of ADSC‑Exos in diabetic wounds by promoting the cellular uptake of exosomes and enhancing angiogenesis.

An H2 S-BMP6 Dual-Loading System with Regulating Yap/Taz and Jun Pathway for Synergistic Critical Limb Ischemia Salvaging Therapy.

Critical limb ischemia, the final course of peripheral artery disease, is characterized by an insufficient supply of blood flow and excessive oxidative stress. H2 S molecular therapy possesses huge potential for accelerating revascularization and scavenging intracellular reactive oxygen species (ROS). Moreover, it is found that BMP6 is the most significantly up-expressed secreted protein-related gene in HUVECs treated with GYY4137, a H2 S donor, based on the transcriptome analysis. Herein, a UIO-66-NH2 @GYY4137@BMP6 co-delivery nanoplatform to strengthen the therapeutic effects of limb ischemia is developed. The established UIO-66-NH2 @GYY4137@BMP6 nanoplatform exerts its proangiogenic and anti-oxidation functions by regulating key pathways. The underlying molecular mechanisms of UIO-66-NH2 @GYY4137@BMP6 dual-loading system lie in the upregulation of phosphorylated YAP/TAZ and Jun to promote HUVECs proliferation and downregulation of phosphorylated p53/p21 to scavenge excessive ROS. Meanwhile, laser-doppler perfusion imaging (LDPI), injury severity evaluation, and histological analysis confirm the excellent therapeutic effects of UIO-66-NH2 @GYY4137@BMP6 in vivo. This work may shed light on the treatment of critical limb ischemia by regulating YAP, Jun, and p53 signaling pathways based on gas-protein synergistic therapy.

Improving Diastolic and Microvascular Function in Heart Transplantation with Donation after Circulatory Death.

The impact of the machine perfusion of donation after circulatory death (DCD) hearts with the novel Custodiol-N solution on diastolic and coronary microvascular dysfunction is unknown. Porcine DCD-hearts were maintained four hours by perfusion with normothermic blood (DCD-B), hypothermic Custodiol (DCD-C), or Custodiol-N (DCD-CN), followed by one hour of reperfusion with fresh blood, including microvascular and contractile evaluation. In another group (DCD group), one hour of reperfusion, including microvascular and contractile evaluation, was performed without a previous maintenance period (all groups N = 5). We measured diastolic function with a balloon catheter and microvascular perfusion by Laser-Doppler-Technology, resulting in Laser-Doppler-Perfusion (LDP). We performed immunohistochemical staining and gene expression analysis. The developed pressure was improved in DCD-C and DCD-CN. The diastolic pressure decrement (DCD-C: -1093 ± 97 mmHg/s; DCD-CN: -1703 ± 329 mmHg/s; DCD-B: -690 ± 97 mmHg/s; p < 0.05) and relative LDP (DCD-CN: 1.42 ± 0.12; DCD-C: 1.11 ± 0.13; DCD-B: 1.22 ± 0.27) were improved only in DCD-CN. In DCD-CN, the expression of eNOS increased, and ICAM and VCAM decreased. Only in DCD-B compared to DCD, the pathways involved in complement and coagulation cascades, focal adhesion, fluid shear stress, and the IL-6 and IL-17 pathways were upregulated. In conclusion, machine perfusion with Custodiol-N improves diastolic and microvascular function and preserves the microvascular endothelium of porcine DCD-hearts.

Exosomal miR-125b-5p derived from adipose-derived mesenchymal stem cells enhance diabetic hindlimb ischemia repair via targeting alkaline ceramidase 2

IntroductionIschemic diseases caused by diabetes continue to pose a major health challenge and effective treatments are in high demand. Mesenchymal stem cells (MSCs) derived exosomes have aroused broad attention as a cell-free treatment for ischemic diseases. However, the efficacy of exosomes from adipose-derived mesenchymal stem cells (ADSC-Exos) in treating diabetic lower limb ischemic injury remains unclear.MethodsExosomes were isolated from ADSCs culture supernatants by differential ultracentrifugation and their effect on C2C12 cells and HUVECs was assessed by EdU, Transwell, and in vitro tube formation assays separately. The recovery of limb function after ADSC-Exos treatment was evaluated by Laser-Doppler perfusion imaging, limb function score, and histological analysis. Subsequently, miRNA sequencing and rescue experiments were performed to figure out the responsible miRNA for the protective role of ADSC-Exos on diabetic hindlimb ischemic injury. Finally, the direct target of miRNA in C2C12 cells was confirmed by bioinformatic analysis and dual-luciferase report gene assay.ResultsADSC-Exos have the potential to promote proliferation and migration of C2C12 cells and to promote HUVECs angiogenesis. In vivo experiments have shown that ADSC-Exos can protect ischemic skeletal muscle, promote the repair of muscle injury, and accelerate vascular regeneration. Combined with bioinformatics analysis, miR-125b-5p may be a key molecule in this process. Transfer of miR-125b-5p into C2C12 cells was able to promote cell proliferation and migration by suppressing ACER2 overexpression.ConclusionThe findings revealed that miR-125b-5p derived from ADSC-Exos may play a critical role in ischemic muscle reparation by targeting ACER2. In conclusion, our study may provide new insights into the potential of ADSC-Exos as a treatment option for diabetic lower limb ischemia.

Cytokine Adsorber Use during DCD Heart Perfusion Counteracts Coronary Microvascular Dysfunction.

Microvascular dysfunction (MVD) in cardiac allografts is associated with an impaired endothelial function in the coronary microvasculature. Ischemia/reperfusion injury (IRI) deteriorates endothelial function. Hearts donated after circulatory death (DCD) are exposed to warm ischemia before initiating ex vivo blood perfusion (BP). The impact of cytokine adsorption during BP to prevent MVD in DCD hearts is unknown. In a porcine DCD model, we assessed the microvascular function of hearts after BP with (DCD-BPCytoS, n = 5) or without (DCD-BP, n = 5) cytokine adsorption (CytoSorb®). Microvascular autoregulation was assessed by increasing the coronary perfusion pressure, while myocardial microcirculation was measured by Laser-Doppler-Perfusion (LDP). We analyzed the immunoreactivity of arteriolar oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal (HNE), endothelial injury indicating cell adhesion molecules CD54, CD106 and CD31, and eNOS. We profiled the concentration of 13 cytokines in the perfusate. The expression of 84 genes was determined and analyzed using machine learning and decision trees. Non-DCD hearts served as a control for the gene expression analysis. Compared to DCD-BP, relative LDP was improved in the DCD-BPCytoS group (1.51 ± 0.17 vs. 1.08 ± 0.17). Several pro- and anti-inflammatory cytokines were reduced in the DCD-BPCytoS group. The expression of eNOS significantly increased, and the expression of nitrotyrosine, HNE, CD54, CD106, and CD31, markers of endothelial injury, majorly decreased in the DCD-BPCytoS group. Three genes allowed exact differentiation between groups; regulation of HIF1A enabled differentiation between perfusion (DCD-BP, DCD-BPCytoS) and non-perfusion groups. CAV1 allowed differentiation between BP and BPCytoS. The use of a cytokine adsorption device during BP counteracts preload-dependent MVD and preserves the microvascular endothelium by preventing oxidative stress and IRI of coronary arterioles of DCD hearts.

Free flaps monitoring by Laser-Doppler Flowmetry in head and neck surgery

Early recognition of free flap vascular impairment is essential for flap salvage attempts. Several methods for surveillance of post-operative flaps are available. Among these, we have extensively used Laser-Doppler Perfusion Flowmetry (LDF) monitoring. We report our experience on this topic and illustrate the advantages and weak points. Over seven years, 110 consecutive free flaps for head and neck reconstruction were monitored using the Periflux System 5000® (Perimed AB, Järfälla, Sweden). In addition to maximum and minimum peaks, a pattern called vasomotion can be detected. Monitoring time lasted from 3 to 7 days, 24/24 h. Six of 110 (5.5%) cases of vascular problems were detected and clinically confirmed. In 5 cases, venous thrombosis was present: 4 patients were successfully treated. In 1 case, both arterial and venous thrombosis occurred. Flowmetry data always showed a more or less sudden disappearance of vasomotion. LDF is a highly sensible, specific and reliable method. It is easy to use and interpret at low cost. Remote monitoring could also be developed.

Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator Mediates the Angiogenic Response to Peripheral Ischemia in Mice With Type 2 Diabetes Mellitus.

Hypoxia-inducible factors (HIFs) are the master regulators of angiogenesis, a process that is impaired in patients with diabetes mellitus (DM). The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF1β) has been implicated in the development and progression of diabetes. Angiogenesis is driven primarily by endothelial cells (ECs), but both global and EC-specific loss of ARNT-cause are associated with embryonic lethality. Thus, we conducted experiments in a line of mice carrying an inducible, EC-specific ARNT-knockout mutation (ArntΔ EC, ERT2) to determine whether aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. Mice were first fed with a high-fat diet to induce diabetes. ArntΔ EC, ERT2 mice were then adminstrated with oral tamoxifen to disrupt Arnt and peripheral angiogenesis was evaluated by using laser-Doppler perfusion imaging to monitor blood flow after hindlimb ischemia. The ArntΔ EC, ERT2 mice had impaired blood flow recovery under both non-diabetic and diabetic conditions, but the degree of impairment was greater in diabetic animals. In addition, siRNA-mediated knockdown of ARNT activity reduced measurements of tube formation, and cell viability in human umbilical vein endothelial cells (HUVECs) cultured under high-glucose conditions. The ArntΔ EC, ERT2 mutation also reduced measures of cell viability, while increasing the production of reactive oxygen species (ROS) in microvascular endothelial cells (MVECs) isolated from mouse skeletal muscle, and the viability of ArntΔ EC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with an ROS inhibitor. Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production.

Antiangiogenic VEGF165b Regulates Macrophage Polarization via S100A8/S100A9 in Peripheral Artery Disease.

Atherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF165a) and VEGFxxxb (VEGF165b) isoforms with existing literature focusing on VEGF165b's role in inhibiting vascular endothelial growth factor receptor 2-dependent angiogenesis. However, we have recently shown that VEGF165b blocks VEGF-A-induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGF165b has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF165b in regulating macrophage function in PAD. Femoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGF165b in regulating macrophage phenotype and function in PAD. First, we found increased VEGF165b expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre clinical PAD models, and adoptive transfer of VEGF165b-expressing bone marrow-derived macrophages or VEGFR1+/- bone marrow-derived macrophages (M1-like phenotype), we demonstrate that VEGF165b inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1+/+ versus hypoxia serum starvation-VEGFR1+/- bone marrow-derived macrophages. In our current study, we demonstrate that increased VEGF165b expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF165b results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.

Critical Ischemia Time, Perfusion, and Drainage Function of Vascularized Lymph Nodes.

Vascularized lymph node transfer is a promising surgical treatment for lymphedema. This study investigated the effect of ischemia on the lymphatic drainage efficiency of vascularized lymph node flaps and the critical ischemia time of lymph nodes. Twenty-four lymph nodes containing groin flaps in 12 Sprague-Dawley rats were dissected. Clamping of the vascular pedicle was performed for 0, 1, 3, 5, 6, or 7 hours; then, each was allowed to reperfuse by means of the vascular pedicle for 1 hour. Perfusion and ischemic changes were assessed using indocyanine green lymphography; laser Doppler flowmetry; and histologic studies with associated lymphatic vessel endothelial hyaluronan receptor-1, CD68, 4',6-diamidino-2-phenylindole, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and glutathione assay stains. The mean latency period of the groin lymph node flaps was 247 ± 67, 83 ± 15, 72 ± 42, 30 ± 18, and 245 ± 85 seconds in the 0-, 1-, 3-, 5-, and 6-hour groups, respectively. Perfusion detected by laser Doppler was 85.2 ± 14.5, 87.2 ± 36.7, 129.8 ± 33.7, 140.4 ± 148.5, 156.1 ± 91.4, and 41.2 ± 34.8 perfusion units at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Cell damage measured by glutathione was 46.8 ± 10.2, 67.7 ± 14.2, 62.8 ± 15.4, 126.6 ± 5.9, 259.0 ± 70.3, and 109.1 ± 27.5 at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Histologically, as ischemia time increased, hemorrhage and congestion became more severe. The critical ischemia time of vascularized lymph nodes is 5 hours in the rodent animal model, verified by indocyanine green lymphatic fluid uptake, laser Doppler perfusion, and histologic assessments. Interestingly, lymphatic drainage and perfusion of vascularized lymph nodes were improved with an increased ischemia time before the critical 5 hours was reached.

Microvascular in-vivo Analysis of Retrograde Venous Arterialization of Ischemic Skeletal Muscle

Objective: Nearly 20% of patients with critical limb ischemia will not be suitable for arterial bypass due to distal small vessel occlusion, and venous arterialization of the distal venous bed might be a valuable surgical option. This study demonstrates the in-vivo microcirculatory effects of this type of intervention. Methods: Using intravital video microscopy, the authors studied the distal skeletal microcirculatory characteristics following venous arterialization of critical hindlimb ischemia in the rat. 25 Wistar rats underwent proximal ligation of the femoral arteries followed by venous arterialization carried out by anastomosing the saphenous vein to the femoral artery using microsurgery techniques. Microcirculatory hemodynamic conditions of the soleus muscle were observed under normal, ischemic, and arterialized conditions. Fluorescein-labeled red cells were used to measure red cell velocities (Vrbc) at the capillaries, and acridine orange injections used to stain endothelial cell nuclei to measure microcirculatory diameters, and leukocyte nuclei to measure leukocyte adhesion. Laser Doppler Perfusion (LDP) units at the distal limb were measured continuously throughout the procedure. Results: Proximal femoral arterial ligation resulted in drastic reductions in LDP and Vrbc. Following distal venous arterialization, LDP returned to an average of 41% of baseline. Vrbc returned to near baseline values in 70% of the capillaries. Flow at the capillary and venular system showed frequent reversals and great variations in velocities. Venules and venu-venular anastomoses diameters increased by 50%. There was immediate macromolecular tracer leakage and leukocyte activation was significantly increased in both ischemic and arterialized groups (15 cells vs 156 and 178 cells respectively). Conclusion: Venous arterialization may provide an improvement in microcirculatory velocities but is accompanied by microcirculatory injury and dysfunction in the acute phase. These results suggest that mechanisms besides microcirculatory hemodynamics play a role in the overall picture of clinical effectivity of the procedure. Key words: Retrograde venous arterialization, ischemic skeletal muscle

Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan sulphate

Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF165) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF165. Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF165 against thermal and enzyme degradation in vitro, and isolated VEGF165 from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n = 8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.

Cutaneous microcirculation during operations with a cardiopulmonary bypass.

Cutaneous microcirculation (cMC) is influenced by many factors. In cardiac surgery, most operations are performed with a cardiopulmonary bypass (CPB) and cardiac arrest induced by cardioplegic solutions. Aim of this study was to examine a correlation between cMC and hemodynamic parameters in patients undergoing heart surgery with two different cardioplegic solutions. 20 patients were included and divided into Histidine-Tryptophane-α-Ketoglutarate solution- (HTK, n = 10) and blood cardioplegia- (BCP, n = 10) groups. With initiation of CPB, cMC was continuously monitored with Laser-Doppler-Perfusion (LDP) until termination of CPB. Additionally, we measured hemoglobin-concentration (HbC) with a Blood-Parameter-Monitoring-System. LDP pulsation was almost equal before and after CPB and decreased during aortic cross clamping. The following factors influenced LDP: central venous pressure (CVP), mean arterial pressure (MAP), total peripheral resistance (TPR) and flow of the heart-lung machine. We measured relative LDP and HbC (RLDP; RHbC). Five and 25 min after administration of cardioplegia, RLDP (1.22±0.8; 1.17±0.94) and RHbC (0.92±0.06; 0.96±0.09) in the HTK-group were lower than in the BCP-group: RLDP (1.58±1.11; 1.58±2.2) and RHbC (1.00±0.05; 0.99±0.13). HTK-patients with a body surface area (BSA) <2 m2 showed a lower RLDP (0.75±0.50), than patients over 2 m2 (RLDP = 1.64±0.97). The cMC is influenced by CPB. Cutaneous LDP monitoring is a non-invasive method, for estimating hemodynamics intraoperatively.

Abstract 008: Perivascular Cell-specific Knockout of the Stem Cell Pluripotency Gene Oct4 Inhibits Angiogenesis in Part by Attenuating Perivascular and Endothelial Cell Migration

Objective: Angiogenesis requires coordinated migration of endothelial cells (EC) and perivascular cells, including smooth muscle cells and pericytes (SMC-P). Perivascular cell-specific mechanisms by which SMC-P migrate and invest EC remain largely unknown. Herein, we used Myh11-CreER T2 SMC-P lineage tracing, combined with SMC-P specific knockout of the stem cell pluripotency gene Oct4, to test the hypothesis that SMC-P derived Oct4 regulates perivascular cell migration and recruitment necessary for angiogenesis. Methods and Results: Myh11-CreER T2 ROSA floxed STOP eYFP Oct4 WT/WT and Myh11-CreER T2 ROSA floxed STOP eYFP Oct4 FL/FL littermate mice were injected with tamoxifen from 6-8 weeks of age to permanently label Myh11-expressing cells with eYFP, without or with Oct4 KO, respectively. Mice were subjected to either corneal alkali burn or hindlimb ischemia (HLI), monitored by live confocal imaging and laser doppler perfusion respectively, and sacrificed for tissue analysis. SMC-P Oct4 KO resulted in markedly impaired eYFP+ (SMC-P derived) migration and increased vascular leak following corneal alkali burn. EC neovascular area and migration distance were also significantly decreased in SMC-P specific Oct4 KO mice. Following HLI, SMC-P Oct4 KO mice had impaired perfusion recovery and decreased capillary density. Slit3 was expressed in eYFP+ cells of the microvasculature in both cornea and muscle and was downregulated following Oct4 KO. RNA-seq and qRT-PCR analysis of cultured SMC revealed dysregulation of Slit3 as well as additional members of the Slit-Robo pathway of guidance genes, including downregulation of the receptors Robo1 and Robo2 and upregulation of Slit2 following loss of Oct4. Conclusions: Taken together, we demonstrate that SMC-P derived Oct4 is essential for angiogenesis in both corneal alkali burn and HLI models. These effects are at least partially due to Oct4-dependent regulation of the Slit-Robo pathway in SMC-P, with Oct4 KO resulting in dysregulated migration of both EC and SMC-P. To our knowledge, this is the first direct evidence that loss of a single gene exclusively in SMC-P impacts angiogenesis following injury.

Ponatinib Exerts Multiple Effects on Vascular Endothelial Cells: Possible Mechanisms and Explanations for the Adverse Vascular Events Seen in CML Patients Treated with Ponatinib

The potent BCR-ABL1-targeting tyrosine kinase inhibitor (TKI) ponatinib is used for the treatment of patients with drug-resistant chronic myeloid leukemia (CML). However, an increased risk of development of cardiovascular events has been described in CML patients treated with ponatinib. The etiology of these adverse events is currently unknown. In an attempt to discover mechanisms underlying ponatinib-induced adverse vascular events, we have evaluated the effects of ponatinib in vitro on human vascular endothelial cells and on contraction of explanted mice aortic rings. In addition, we examined the effects of ponatinib on angiogenesis in vivo in a mouse model of hind limb ischemia. Ponatinib dose-dependently induced apoptosis in human coronary artery endothelial cells (HCAEC) in a caspase assay (relative apoptosis vs. 1% DMSO: ponatinib 50 nM: 1.79±0.38, p<0.001; ponatinib 100 nM: 2.13±0.42, p<0.001) and this drug effect could partially be blocked by addition of insulin (ponatinib 100 nM + insulin 5 µg/ml: 1.70±0.18, p<0.05). In addition, ponatinib was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and the human microvascular endothelial cell line HMEC-1, with IC50 values ranging between 100 and 250 nM (p<0.05) as determined by thymidine-incorporation assay. Using a phospho-receptor tyrosine kinase assay in HCAEC, we found that ponatinib also inhibits fetal bovine serum-induced phosphorylation of the VEGF receptor KDR as well as phosphorylation of MER and insulin receptors, which play a role in angiogenesis, vascular homeostasis, and vessel protection. We also found that ponatinib (1 µM, 4 hours) increases adhesion of HUVEC to a plastic-surface compared to DMSO control (Figure). Based on clinical observations of vasoconstriction in ponatinib-treated patients, we also applied ponatinib on aortic rings harvested from C57BL/6 mice. Ponatinib (100 nM, overnight) enhanced norepinephrine-induced vasoconstriction (log EC50: control -7.76±0.06 vs. ponatinib -7.96±0.05, p<0.05, n=6) and inhibited acetylcholine-mediated vasodilatation (log IC50: control -7.45±0.05 vs. ponatinib -7.06±0.1, p<0.001) as shown by myography. These drug effects were blocked by inhibition of nitric oxide (using nitric oxide synthase inhibitor L-NNA, 100 µM) or COX (by applying diclofenac, 3 mg/l), suggesting that ponatinib promotes the generation of vasoconstricting prostanoids. Ponatinib effects were also blocked by the calcium channel blocker nifedipine (1 µM). In C57BL/6 mice, ponatinib (5 mg/kg/day for 35 days) was found to inhibit blood flow recovery in a hind limb ischemia model as shown by Laser-Doppler perfusion imaging after femoral artery ligation. The blood perfusion ratios of the ischemic limb vs. non-ischemic limb at week 5 were: control group: 0.67±0.07 vs. ponatinib: 0.56±0.1; p<0.05). Ponatinib-treated mice also developed toe and foot necrosis more frequently than control mice (necrosis score: control: 0.3 vs. ponatinib: 1.3). In summary, ponatinib affects endothelial cell growth and vasomotor function in-vitro as well as blood flow recovery in a mouse model. These findings might help explain the occurrence of vascular events in CML patients treated with ponatinib and may lead to development of therapeutic strategies for prevention and treatment of ponatinib-induced adverse events. [Display omitted] DisclosuresKirchmair:Ariad: Research Funding. Valent:Ariad: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Abstract 222: N-Acetylcysteine Enhances Amputation Stump Healing and Perfusion in Diabetes

Over 150,000 extremity amputations are performed in the US per year. More than 60% of these are performed in patients with diabetes and peripheral arterial disease, and 25% require subsequent re-amputation due to poor surgical site healing. The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood, which greatly limits management options. N-acetylcysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, promotes endothelial cell function and angiogenesis, and is proposed to have therapeutic benefits in the setting of diabetes. To test the hypothesis that NAC can improve surgical site healing in chronically ischemic amputation stumps in the setting of diabetes, we developed a novel in vivo murine hindlimb ischemia-amputation model. In this model, streptozotocin-treated C57B6 mice are allowed to develop hyperglycemia for 1 month, followed by unilateral hindlimb femoral artery ligation. After 1 week, the recovered yet still mal-perfused hindlimb undergoes an infrageniculate amputation. Compared to sham control, mice receiving a daily intraperitoneal administration of NAC 150mg/kg demonstrated a 31% improvement in stump healing (P=0.06), 160% increase in hindlimb stump laser-Doppler perfusion at 3 days post-amputation (P=0.03), as well as 179% increase in hindlimb adductor muscle neovascularization (P=0.01). Adductor muscle segments from ischemic amputated hindlimbs also demonstrated less muscle damage, and expressed higher levels of de-palmitoylated activated Gaq (P&lt;0.05), which is essential for ischemia-induced neovascularization. Similarly, HUVECs treated with NAC 3mM demonstrated a transient 19% increase in de-palmitoylated Gaq (P=0.03), suggesting a novel mechanism of action for NAC. These findings demonstrate that NAC may have important therapeutic benefits in amputation stump healing in the setting of diabetes, which may translate into a valuable treatment option in vulnerable patient populations.

Influence of forearm muscle metaboreceptor activation on sweating and cutaneous vascular responses during dynamic exercise.

We examined whether the sustained activation of metaboreceptor in forearm during cycling exercise can modulate sweating and cutaneous vasodilation. On separate days, 12 young participants performed a 1.5-min isometric handgrip exercise at 40% maximal voluntary contraction followed by 1) 9-min forearm ischemia (Occlusion, to activate metaboreceptor) or 2) no ischemia (Control) in thermoneutral conditions (27°C, 50%) with mean skin temperature clamped at 34°C. Thirty seconds after the handgrip exercise, participants cycled for 13.5 min at 40% V̇o2 max For Occlusion, forearm ischemia was maintained for 9 min followed by no ischemia thereafter. Local sweat rate (SR, ventilated capsule) and cutaneous vascular conductance (CVC, laser-Doppler perfusion units/mean arterial pressure) on the contralateral nonischemic arm as well as esophageal and skin temperatures were measured continuously. The period of ischemia in the early stages of exercise increased SR (+0.03 mg·cm(-2)·min(-1), P < 0.05) but not CVC (P > 0.05) above Control levels. No differences were measured in the esophageal temperature at which onset of sweating (Control 37.19 ± 0.09 vs. Occlusion 37.07 ± 0.09°C) or CVC (Control 37.21 ± 0.08 vs. Occlusion 37.08 ± 0.10°C) as well as slopes for these responses (all P > 0.05). However, a greater elevation in SR occurred thereafter such that SR was significantly elevated at the end of the ischemic period relative to Control (0.37 ± 0.05 vs. 0.23 ± 0.05 mg·cm(-2)·min(-1), respectively, P < 0.05) despite no differences in esophageal temperature. We conclude that the activation of forearm muscle metaboreceptor can modulate sweating, but not CVC, during cycling exercise without affecting the core temperature-SR relationship.

A Role for Cyclooxygenase and Nitric Oxide Synthase in Sweating and Skin Blood Flow During Exercise in the Heat in Young Individuals with Type 1 Diabetes

Recent studies show that individuals with type 1 diabetes mellitus (T1DM) have impaired heat loss responses. This is evidenced as a decrease in local sweat rate and an increase in core body temperature during exercise in the heat compared to non‐diabetic matches; however, the underlying mechanisms are currently unknown. Previous studies have shown that nitric oxide synthase (NOS) and cyclooxygenase (COX) increase sweating, while NOS, but not COX, contributes to cutaneous vasodilation in young adults exercising in the heat. Thus, the aim of our study was to determine if the impairments in local heat loss responses displayed by individuals with T1DM may be attributed to COX and/or NOS activity. Seven (5 males, 2 females) young (24±3 years) individuals with T1DM were instrumented with four microdialysis fibres in the forearm skin which were perfused with either 1) 10 mM ketorolac (KETO, non‐selective COX inhibitor), 2) 10 mM NG‐nitro‐L‐arginine methyl ester (L‐NAME, non‐selective NOS inhibitor), 3) a combination of L‐NAME and KETO (L‐NAME+KETO), or 4) lactated Ringer (Control). Sweat rate (ventilated capsule) and cutaneous vascular conductance (CVC; laser Doppler perfusion units/mean arterial pressure) were measured continuously throughout the protocol. Participants performed two 30 min bouts of exercise at a fixed rate of heat production of 400 W in the heat (35°C, 20% relative humidity); separated by a 20‐ and 40‐min recovery respectively. During the first and second exercise bouts, we showed that the inhibition of COX (KETO) resulted in an increase in sweat rate relative to Control (average of last 5 min of second exercise; Control: 1.02±0.11 mg min−1 cm−2, KETO: 1.12±0.12 mg min−1 cm−2; P=0.029). However, a decrease in sweat rate was observed when both NOS and COX were inhibited (L‐NAME+KETO: 0.86±0.11 mg min−1 cm−2; P=0.031). Furthermore, inhibition of NOS alone did not significantly modulate sweating (L‐NAME: 0.93±0.11 mg min−1 cm−2; P=0.11). With respect to CVC, the inhibition of NOS (average of last 5 min of second exercise, 45±9%max), as well as NOS and COX (49±4%max) resulted in similar decreases in CVC relative to Control (68±1%max)(both P&lt;0.05). In contrast, the inhibition of COX alone did not modulate CVC (64±4%max; P=0.33). Our results demonstrate that COX attenuates sweating during exercise in the heat in young adults with T1DM, which may occur through NOS‐dependent mechanisms. Finally, we show that individuals with T1DM exhibit NOS‐ but not COX‐dependent cutaneous vasodilation during exercise in the heat.Support or Funding InformationThis study was supported by grants from the Canadian Institutes of Health Research (286363; funds held by Dr. Glen P. Kenny).

A shape-controlled tunable microgel cell delivery platform for low-dose delivery of primed stem cells for in vivo therapeutic neovascularization

Event Abstract Back to Event A shape-controlled tunable microgel cell delivery platform for low-dose delivery of primed stem cells for in vivo therapeutic neovascularization Dilip Thomas1, 2, Grazia Marsico1, Arun Thirumaran2, Bartlomiej Lukasz3, Kerry Thompson4, Peter Dockery4, Brian Rodriguez3, Martina Marchetti-Deschmann5, Timothy O'Brien1, 2 and Abhay Pandit1 1 National University of Ireland Galway, CÚRAM, Ireland 2 National University of Ireland, REMEDI, Ireland 3 University College Dublin, Conway Institute, Ireland 4 National University of Ireland Galway, Anatomy, Ireland 5 Vienna University of Technology, Institute of Chemical Technologies and Analytics, Austria Introduction: Hydrogel-based cell delivery platforms fabricated from synthetic or natural polymers have evolved from a cytoprotective design to a functional biomaterial design, which not only offers better cell survival but also modulates cellular behaviour via intrinsic biophysical or biochemical cues. Tissue regenerative processes are driven by reparative mesenchymal stem cells (MSCs) under the influence of biochemical cues dictated by the microenvironment. Prolonged ischemia and lack of blood supply drive cellular apoptosis and tissue death. However, delivery of primed stem cells via a tunable microenvironment triggers an ‘angiocrine’ response, driving therapeutic angiogenesis[1]. Hence, in this study, it is hypothesised that delivery of primed human mesenchymal stem cells on a shape-controlled microgel platform at a low-cell dose promotes angiogenesis in a murine model of hind-limb ischemia (HLI). The specific objectives of the study were to investigate the cellular behaviour on tunable parameters such as matrix concentration, cross-linker concentration and cell densities and therapeutic efficacy of the optimized group in a hind-limb ischemia model. Materials and Methods: Collagen-based microgels of concentrations 1, 2 and 3mg/ml were fabricated by neutralizing type-I collagen and dispensing a mixture composed of cells and cross-linker 4S-Star-PEG Mw 10000 KDa (Jenkem Tech, U.S.A) on to a hydrophobic surface. hMSCs at an optimized cell density of 0.8x106 were embedded within the microgels[2]. Cell embedded microgels were tested for changes in morphology by confocal microscopy; protein and gene expression quantitation by multiplex ELISA and gene arrays; and changes in matrix stiffness with atomic force microscopy and confocal microscopy. In vivo experiments were performed in a Balb/c nude hind-limb ischemia mouse model. Animal groups (n=12/group) were PBS; microgels alone; microgels with 50,000 hMSCs; 50,000 and 1,000,000 cells alone. Laser-Doppler perfusion and clinical signs of disease severity were assessed along with molecular evaluation using; multiplex ELISA, gene expression arrays and MALDI-imaging mass spectrometry. Statistical analysis was performed using two-way ANOVA with p<0.05. Results and Discussion: The use of 4S-StarPEG combined with the high surface hydrophobicity allowed gelation after 40 minutes, conferring a spherical shape to the microgels. Over 80% viability and low apoptosis was observed using flow-cytometry. 2mg/ml microgels at 0.8x106 cell density showed up-regulation of paracrine factors involved in angiogenesis and distinguishing integrin expression profile (Figure1). AFM analysis revealed insignificant changes in matrix stiffness and unique cell morphology in 2mg/ml microgels compared to 1 and 3mg/ml microgels. Results from the in vivo hind-limb ischemia studies (Figure 2) showed increased blood perfusion, up-regulation of protein and gene markers related to angiogenesis, and changes in glycan environment between the treatment and control groups. Conclusion: Tunable shape-controlled collagen based microgel platform can be used to deliver hMSCs at a low cell dose for angiogenesis in vivo. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073. Science Foundation Ireland (SFI) under grant no.09/SRC/B1794.