Ischemic Flap Model Research Articles

The effect of pitavastatin in an ischemic skin flap model in rats

The effect of pitavastatin in an ischemic skin flap model in rats

Bioactive extracellular matrix scaffolds engineered with proangiogenic proteoglycan mimetics and loaded with endothelial progenitor cells promote neovascularization and diabetic wound healing

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

Effect of natural hirudin on revascularization of ischemic skin flaps in rats by Micro-CT

To investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction. Thirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction. Both groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) ( t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group ( P<0.05). Natural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.

Perioperative Treatment with a Prolyl Hydroxylase Inhibitor Reduces Necrosis in a Rat Ischemic Skin Flap Model.

The hypoxia-inducible factor (HIF) pathway, regulated by prolyl hydroxylase, is central to tissue adaptation to ischemia. The authors tested whether the prolyl hydroxylase inhibitor dimethyloxalylglycine reduces skin flap necrosis. Dorsal skin flaps were raised on hairless rats, with dimethyloxalylglycine delivered intraperitoneally and/or topically for 7 days before and after surgery. After 14 treatment days, percentage of flap necrosis was compared grossly and tissue perfusion compared with an in vivo imaging system. Angiogenesis was compared using immunohistochemical CD31 staining and enzyme-linked immunosorbent assay for tissue vascular endothelial growth factor. Expression levels of HIF-1α and terminal deoxynucleotidyl transferase-mediated dUDP end-labeling were compared using immunohistochemical staining. Complete blood counts and gross necropsy specimens were obtained to assess systemic toxicity. Dimethyloxalylglycine administration significantly improved postoperative flap viability, with combined topical and intraperitoneal dimethyloxalylglycine administration leading to reduced necrosis on postsurgical day 7 at 6 mg/kg/day, 12 mg/kg/day, 24 mg/kg/day, and 48 mg/kg/day versus controls (all p < 0.05). Compared with controls (unperfused, 39.9 ± 3.8 percent), dimethyloxalylglycine treatment led to a dose-dependent decrease in unperfused tissue at 6 mg/kg/day (11.4 ± 1.7 percent), 12 mg/kg/day (9.4 ± 4.2 percent), 24 mg/kg/day (4.7 ± 2.6 percent), and 48 mg/kg/day (1.4 ± 0.9 percent) (all p < 0.001). Topical dimethyloxalylglycine application alone administered at 48 mg/kg/day was sufficient to improve flap viability (p = 0.005). Dimethyloxalylglycine-treated flaps exhibited higher CD31 staining (p = 0.004), tissue vascular endothelial growth factor (p = 0.007), HIF-1α staining (p < 0.001), and reduced terminal deoxynucleotidyl transferase-mediated dUDP end-labeling staining (p = 0.045). There were no differences in hematocrit or macroscopic organ changes on gross necropsy. Topical and systemic targeting of the HIF-1 pathway may be a promising therapeutic approach to improve flap resistance to ischemia.

Discussion: Perioperative Treatment with a Prolyl Hydroxylase Inhibitor Reduces Necrosis in a Rat Ischemic Skin Flap Model.

Discussion: Perioperative Treatment with a Prolyl Hydroxylase Inhibitor Reduces Necrosis in a Rat Ischemic Skin Flap Model.

Oxygen-Releasing Antioxidant Cryogel Scaffolds with Sustained Oxygen Delivery for Tissue Engineering Applications.

With the advancement in biomaterial sciences, tissue-engineered scaffolds are developing as a promising strategy for the regeneration of damaged tissues. However, only a few of these scaffolds have been translated into clinical applications. One of the primary drawbacks of the existing scaffolds is the lack of adequate oxygen supply within the scaffolds. Oxygen-producing biomaterials have been developed as an alternate strategy but are faced with two major concerns. One is the control of the rate of oxygen generation, and the other is the production of reactive oxygen species (ROS). To address these concerns, here, we report the development of an oxygen-releasing antioxidant polymeric cryogel scaffold (PUAO-CPO) for sustained oxygen delivery. PUAO-CPO scaffold was fabricated using the cryogelation technique by the incorporation of calcium peroxide (CPO) in the antioxidant polyurethane (PUAO) scaffolds. The PUAO-CPO cryogels attenuated the ROS and showed a sustained release of oxygen over a period of 10 days. An in vitro analysis of the PUAO-CPO cryogels showed their ability to sustain H9C2 cardiomyoblast cells under hypoxic conditions, with cell viability being significantly better than the normal polyurethane (PU) scaffolds. Furthermore, in vivo studies using an ischemic flap model showed the ability of the oxygen-releasing cryogel scaffolds to prevent tissue necrosis upto 9 days. Histological examination indicated the maintenance of tissue architecture and collagen content, whereas immunostaining for proliferating cell nuclear antigen confirmed the viability of the ischemic tissue with oxygen delivery. Our study demonstrated an advanced approach for the development of oxygen-releasing biomaterials with sustained oxygen delivery as well as attenuated production of residual ROS and free radicals because of ischemia or oxygen generation. Hence, the oxygen-releasing PUAO-CPO cryogel scaffolds may be used with cell-based therapeutic approaches for the regeneration of damaged tissue, particularly with ischemic conditions such as myocardial infarction and chronic wound healing.

* A Rodent Excision Model for Ischemia-Impaired Wound Healing.

Delayed wound healing and the potentially resulting chronic wounds are a challenging clinical problem. Available therapeutic strategies are limited in both number and efficacy. For developing and establishing novel treatment approaches appropriate clinically relevant animal models are essential. The aim of the study was to establish a reliable and reproducible delayed wound healing model, which simulates the clinical scenario of compromised vascular tissue perfusion (hypoxia/ischemia). Therefore a standard rodent ischemic flap model was modified by challenging the tissue with ascending degrees of ischemia using different surgical approaches (minimal, mild, moderate, and severe ischemic invasive approach). Then a full-thickness circular wound was excised in both the non-/hypoperfused flap area and in the normally perfused contralateral region serving as an internal control. Wound healing progress was compared. Superficial tissue perfusion was measured by Laser Doppler imaging technique, which showed persistent ischemia in the moderate and severe invasive surgical approaches 7 days after wounding. Wound closure assessed by planimetric analysis occurred significantly slower in the ischemic wounds compared to the contralateral nonischemic wounds in the moderate invasive approach. Histologic evaluations in this approach showed signs of tissue necrosis and impaired angiogenesis in the ischemic wounds. Therefore, it can be concluded that this clinically relevant animal model is suitable to study mechanism in ischemia-impaired wound healing. Furthermore, it allows evaluating the efficacy of therapeutic strategies for impaired wound healing and comparing the results with an internal control wound.

Abstract: Topical Application of Nitrosonifedipine, a Novel Free Radical Scavenger, Ameliorate the Ischemic Skin Flap Necrosis

INTRODUCTION: Ischemic flap necrosis is often occurred by insufficient blood supply, which prolongs the treatment period and occasionally requires an additional surgery. Ischemic flaps generate an excess amount of free radicals which is regarded as a major factor of ischemic skin necrosis. Thus free-radical scavengers would be an effective drugs in improvement of flap survival. In our previous studies, we have reported nitrosonifedipine (NO-NIF), which is a photolytic compound of nifedipine, possesses a potent radical scavenging activity, and shows the favorable effects against vascular endothelial dysfunction and type 2 diabetic nephropathy. In this study, we evaluated the ameliorating effect of NO-NIF on the ischemic flap model mice. MATERIAL AND METHODS: 9–10 weeks old Male C57BL/6 mice were divided into 2 groups, NO-NIF or control (n=6 in each group), respectively. A 1.0 × 3.0 cm cranially based random pattern flap was elevated on the dorsum of mice. NO-NIF 30 mg/kg or vehicle was injected subcutaneously immediate after the operation and once a day until evaluation. Seven days after surgery, the survival area was calculated as a percentage of the total flap area. To detect the oxidative stress, malondialdehyde (MDA) in the distal part of the flap at post-operative day 1 and 3 was measured by thiobarbituric acid reactive substances assay. Protein expression of p22phox, an essential component of NADPH-oxidase, in the flap was measured by western blotting. RESULTS: At post-operative day 7, the flap survival area was significantly larger in the NO-NIF-treated mice than controls (78.29 ± 7.04% vs. 51.81 ± 6.85%, p=0.021). The amount of MDA significantly decreased in the NO-NIF-treated mice at post-operative day 3 (2.31 ± 0.28µmol/g protein vs. 4.21 ± 0.32µmol/g protein, p=0.001), whereas MDA was same level in the both groups at the post-operative day 1 (2.77 ± 0.61µmol/g protein vs. 2.96 ± 0.51µmol/g protein). In a manner consistent with MDA levels, protein expression level of p22phox was decreased in the NO-NIF-treated mice at post-operative day 3 (p=0.002). CONCLUSIONS: We present the ameliorating effect of NO-NIF on ischemic flap survival. MDA and p22phox protein was decreased by NO-NIF treatment, which suggests the ameliorating effect was exerted via free radical scavenging. This investigation indicates that free-radical scavengers including NO-NIF are effective drugs in improvement of flap survival. DISCLOSURE/FINANCIAL SUPPORT:None.

The Effect of Red Ginseng Extract Intake on Ischemic Flaps

ABSTRACTRed ginseng is well known for its angiogenic effects and its effect of increasing expression of vascular endothelial growth factors (VEGFs), but little experimental evidence has been published. In this study, we examined the effect of red ginseng using an ischemic flap model. Twenty male Sprague-Dawley rats were divided into two groups of 10. One group drank red ginseng solution from 7 days prior to surgery to 7 days after, whereas the other group drank distilled water. We created a local flap on the back of each rat. We analyzed the surviving area of the flap for 10 days after surgery and measured the blood flow of the flap. Ten days after the operation, CD31-positive vessels and VEGF expression were examined by immunohistochemistry. The percentages of surviving areas of the flap were 76 ± 3% for the experimental group and 39 ± 5% for the control group (P = 0.0002). Blood flow in the experimental group increased for 10 days after the surgery. The number of newly generated capillaries in the experimental group was 14.0 ± 3.5, which was significantly higher than 5.7 ± 1.9 in the control group. The expression of VEGF in the experimental group was significantly higher than in the control group (p = 0.0003). Administration of red ginseng extract increases the survival of ischemic flaps via angiogenesis and elevated blood flow. Further clinical studies are warranted to apply the effect shown in this current investigation to various ischemic conditions.

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis

Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration. Statement of significanceIn our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

Demonstration of the rat ischemic skin wound model.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

A dissolved oxygen dressing: a pilot study in an ischemic skin flap model.

To determine if skin flap failure rates could be improved with the use of a dissolved oxygen wound dressing in a porcine model. Full-thickness skin flaps (4 × 16 cm) were raised on pigs. Flaps were randomly assigned after surgery to experimental treatment with a dissolved oxygen dressing (treatment group) or a hydrogel dressing (control group). Flaps were evaluated daily for 14 days. Skin flaps that failed any one of four key clinical outcomes were considered failures. Histological parameters (including skin and subcutaneous necrosis, inflammation, ischemia, fibrosis, and bacterial load) were compared by a blinded histopathologist. Sixteen full-thickness skin flaps were raised on four pigs. All animals survived surgery and all incisions were evaluable. Clinical flap failure was observed in six (75%) control-treated wounds and in two (25%) dissolved oxygen-treated wounds. Histological evaluation demonstrated no significant differences in the proximal 75% of the flaps. There were significant differences in a number of histological parameters in the distal 25% in favor of the dissolved oxygen dressing. Flaps treated with a dissolved oxygen dressing had fewer clinical failures and improved histological profiles compared with control-treated flaps, suggesting that increasing local oxygen supply may improve the local wound healing environment.

Tanshinone IIA pretreatment renders free flaps against hypoxic injury through activating Wnt signaling and upregulating stem cell-related biomarkers.

Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.

Tanshinone IIA pretreatment protects free flaps against hypoxic injury by upregulating stem cell-related biomarkers in epithelial skin cells.

BackgroundPartial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability.MethodsPrimary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 weeks. Cell Counting Kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo.ResultsEpidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue.ConclusionsTSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.

Epidermal or Dermal Specific Knockout of PHD-2 Enhances Wound Healing and Minimizes Ischemic Injury

IntroductionHypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.MethodsEpidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.ResultsqRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).ConclusionsPHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.

0362: Interleukin 17 production induced by angiotensin II type 2 receptor activation in T cells drives flow-mediated outward remodeling of mouse resistance arteries

Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=xx per group, P<00.1) in control mice. No remodeling occurred in nude mice and in mice treated with anti-CD3 antibodies. In control arteries lymphocyte T cells accumulate around HF arteries at day 1 (shown using both immunohistology and flow cytometry) and they expressed angiotensin II type 2 receptor (AT2R). In isolated T cells AT2R stimulation induced preferentially IL-17 production (20±6pg/ml to 50±5pg/ml, n=xx per group, P<00.1). We then tested the occurrence of remodeling in mice lacking AT2R or IL-17. In both AT2R-/- and IL-17-/- mice diameter expansion did not occur whereas T cells and myeloid cells accumulation around the HF artery was not affected. IL-17 infusion (5ng/h, Alzet osmotic minipump) to nude mice and to AT2R-/- mice restored HF remodeling to control level. In order to confirm the role of AT2R in flow-mediated remodeling in another vascular territory, neovascularization was examined using an ischemic skin flap model in AT2R-/- mice. As in mesenteric arteries, the absence of AT2R prevented diameter expansion in the arteries feeding the skin flap. we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Effect of endogenous bone marrow derived stem cells induced by AMD-3100 on expanded ischemic flap.

The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.

Abstract 238: Interleukin 17 Production Induced by Angiotensin Ii Type 2 Receptor Activation in T Cells Drives Flow-mediated Outward Remodeling of Mouse Resistance Arteries

Objective: Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. Results: After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=9 per group, P&lt;00.1) in control mice. No remodeling occurred in nude mice and in mice treated with anti-CD3 antibodies. In control arteries lymphocyte T cells accumulate around HF arteries at day 1 (shown using both immunohistology and flow cytometry) and they expressed angiotensin II type 2 receptor (AT2R). In isolated T cells AT2R stimulation induced preferentially IL-17 production (20±6pg/ml to 50±5pg/ml, n=7 per group, P&lt;00.1). We then tested the occurrence of remodeling in mice lacking AT2R or IL-17. In both AT2R-/- and IL-17-/- mice diameter expansion did not occur whereas T cells and myeloid cells accumulation around the HF artery was not affected. IL-17 infusion (5ng/h, Alzet osmotic minipump) to nude mice and to AT2R-/- mice restored HF remodeling to control level. In order to confirm the role of AT2R in flow-mediated remodeling in another vascular territory, neovascularization was examined using an ischemic skin flap model in AT2R-/- mice. As in mesenteric arteries, the absence of AT2R prevented diameter expansion in the arteries feeding the skin flap. Conclusion: we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Developing digital tissue phantoms for hyperspectral imaging of ischemic wounds

Hyperspectral imaging has the potential to achieve high spatial resolution and high functional sensitivity for non-invasive assessment of tissue oxygenation. However, clinical acceptance of hyperspectral imaging in ischemic wound assessment is hampered by its poor reproducibility, low accuracy, and misinterpreted biology. These limitations are partially caused by the lack of a traceable calibration standard. We proposed a digital tissue phantom (DTP) platform for quantitative calibration and performance evaluation of spectral wound imaging devices. The technical feasibility of such a DTP platform was demonstrated by both in vitro and in vivo experiments. The in vitro DTPs were developed based on a liquid blood phantom model. The in vivo DTPs were developed based on a porcine ischemic skin flap model. The DTPs were projected by a Hyperspectral Image Projector (HIP) with high fidelity. A wide-gap 2nd derivative oxygenation algorithm was developed to reconstruct tissue functional parameters from hyperspectral measurements. In this study, we have demonstrated not only the technical feasibility of using DTPs for quantitative calibration, evaluation, and optimization of spectral imaging devices but also its potential for ischemic wound assessment in clinical practice.