Hindlimb Tissue Research Articles

Carcass characteristics and meat quality of thin-tailed Kivircik and fat-tailed Kangal Akkaraman lambs according to EUROP carcass fatness and conformation classes

A carcass classification method is expected to effectively categorize carcasses based on both carcass and meat quality criteria. Localisation of adipose tissue on the carcass differs between thin- and fat-tailed sheep breeds, which may influence the effectiveness of the EUROP classification system in distinguishing lamb carcasses. The aim of the study was to investigate the effects of EUROP carcass fatness and conformation classes on carcass characteristics and meat quality in thin-tailed Kivircik and fat-tailed Kangal Akkaraman lambs. Slaughtering, carcass, and meat quality data of 115 lambs (67 Kivircik and 48 Kangal Akkaraman) were examined. Hierarchical cluster analysis yielded 3 clusters in both breeds. Differences among lamb carcasses in different clusters were significant for nearly all carcass characteristics in both breeds. Fatness and conformation classes significantly influenced on most of the carcass characteristics in both breeds. However, the effect of fatness class on percentages of valuable joints, and the effect of conformation class on hind limb tissue composition were not significant in Kangal Akkaraman lambs. As carcass fatness and conformation classes increased in Kivircik lambs, meat colour became darker. In Kangal Akkaraman lambs, fatness class had no influence on meat colour. The effects of fatness and conformation classes on sensory characteristics were not significant in both breeds, except fatness effect on odour intensity in Kangal Akkaraman lambs. In conclusion, the EUROP classification system may not be as effective in distinguishing carcass and meat quality in Kangal Akkaraman lambs as in the Kivircik breed.

Evaluation of vascular repair by tissue-engineered human acellular vessels or expanded polytetrafluoroethylene grafts in a porcine model of limb ischemia and reperfusion.

This study evaluated performance of a tissue-engineered human acellular vessel (HAV) in a porcine model of acute vascular injury and ischemia. The HAV is an engineered blood vessel consisted of human vascular extracellular matrix proteins. Limb reperfusion and vascular outcomes of the HAV were compared with those from synthetic expanded polytetrafluoroethylene (ePTFE) grafts. Thirty-six pigs were randomly assigned to four treatment groups, receiving either the HAV or a PTFE graft following a hind limb ischemia period of either 0 or 6 hours. All grafts were 3-cm-long interposition 6-mm diameter grafts implanted within the right iliac artery. Animals were not immunosuppressed and followed for up to 28 days after surgery. Assessments performed preoperatively and postoperatively included evaluation of graft patency, hind limb function, and biochemical markers of tissue ischemia or reperfusion injury. Histological analysis was performed on explants to assess host cell responses. Postoperative gait assessment and biochemical analysis confirmed that ischemia and reperfusion injury were caused by 6-hour ischemia, regardless of vascular graft type. Hind limb function and tissue damage biomarkers improved in all groups postoperatively. Final patency rates at postoperative day 28 were higher for HAV than for ePTFE graft in both the 0-hour (HAV, 85.7%; ePTFE, 66.7%) and 6-hour (HAV, 100%; ePTFE, 75%) ischemia groups, but these differences were not statistically significant. Histological analyses identified some intimal hyperplasia and host reactivity to the xenogeneic HAV and also to the synthetic ePTFE graft. Positive host integration and vascular cell infiltration were identified in HAV but not ePTFE explants. Based on the functional performance and the histologic profile of explanted HAVs, this study supports further investigation to evaluate long-term performance of the HAV when used to repair traumatic vascular injuries.

Multimodal imaging distribution assessment of a liposomal antibiotic in an infectious disease model.

Liposomes are promising targeted drug delivery systems with the potential to improve the efficacy and safety profile of certain classes of drugs. Though attractive, there are unique analytical challenges associated with the development of liposomal drugs including human dose prediction given these are multi-component drug delivery systems. In this study, we developed a multimodal imaging approach to provide a comprehensive distribution assessment for an antibacterial drug, GSK2485680, delivered as a liposomal formulation (Lipo680) in a mouse thigh model of bacterial infection to support human dose prediction. Positron emission tomography (PET) imaging was used to track the in vivo biodistribution of Lipo680 over 48h post-injection providing a clear assessment of the uptake in various tissues and, importantly, the selective accumulation at the site of infection. In addition, a pharmacokinetic model was created to evaluate the kinetics of Lipo680 in different tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was then used to quantify the distribution of GSK2485680 and to qualitatively assess the distribution of a liposomal lipid throughout sections of infected and non-infected hindlimb tissues at high spatial resolution. Through the combination of both PET and MALDI IMS, we observed excellent correlation between the Lipo680-radionuclide signal detected by PET with the GSK2485680 and lipid component signals detected by MALDI IMS. This multimodal translational method can reduce drug attrition by generating comprehensive biodistribution profiles of drug delivery systems to provide mechanistic insight and elucidate safety concerns. Liposomal formulations have potential to deliver therapeutics across a broad array of different indications, and this work serves as a template to aid in delivering future liposomal drugs to the clinic.

The gut microbiota regulates acute foreign body reaction and tissue repair after biomaterial implantation

We hypothesized that the host microbiome may influence foreign body responses following biomaterial implantation. To test this, we implanted a variety of clinically relevant biomaterials into germ-free or antibiotic-treated mice. Surprisingly, these mice displayed less fibrous tissue deposition, reduced host cell recruitment to the implant site, and differential expression of angiogenic and inflammatory markers. These observations were reversed upon fecal microbiome reconstitution, confirming a causal role of the host microbiome. In a clinically relevant disease model, microbiome-depleted mice cleared hyaluronic acid and bone marrow mononuclear cells from ischemic hind limb tissues more slowly, resulting in an improved therapeutic response. Findings were confirmed in pigs which showed reduced fibrotic responses to a variety of implanted materials. Lastly, we profiled changes in the host microbiome following material implantation, implicating several key bacteria phyla.

Allometric growth of non-carcass components and hind limb tissue composition in Saanen goat kids reared under natural and artificial systems

The aim was to investigate the growth of certain non-carcass components and hind limb tissues using allometric coefficients in Saanen goat kids reared under natural and artificial systems and slaughtered at 80 days or 120 days of age. 42 Saanen kids were used in the study [(24 artificial reared kids (SA-80: 12 kids, SA-120: 12 kids), 18 natural reared kids (SA-80: 9 kids, SA-120: 9 kids)] for determination of indicated parameters. The effect of rearing system was not significant in weights of non-carcass components and hind limb tissue compositions, which indicates that, even though they were fed with milk replacers, artificial reared kids can have similar growth performance when they slaughtered at similar age with natural raised kids. However, slaughter age had significant effect on many non-carcass components, since the weight of the organs increases in parallel with animal’s live weight. It is seen that allometric growth of non-carcass parameters, except skin and testis in both rearing groups and stomach and intestines in natural rearing, was slower (b

30. The Effect of Lymphatic Proliferation in Extremity Trauma And Aberrant Wound Healing

Purpose: Traumatic injury results in alterations to tissue organization requiring a highly orchestrated process for proper healing. Following traumatic injury, muscle, tendon, vasculature, nerves, and bone are destroyed resulting in impaired regeneration. There is a paucity of studies investigating the role of lymphatic vasculature network plays in tissue healing following traumatic injury. This is surprising considering the role that the lymphatic system plays in the normal inflammatory response and managing resulting edema which is critical in preventing infection and initiating the healing cascade. Alterations in this process can result in aberrant healing seen following traumatic injury. Utilizing a reliable and reproducible method of traumatic injury, we investigated the role of lymphatic vessels in and aberrant wound healing utilizing novel bioinformatic techniques and 2- and 3-dimensional confocal imaging modalities. Methods: We utilized a murine model of traumatic injury resulting in formation of heterotopic ossification (HO). We performed a 30% total body surface area burn to the dorsum of the mouse and a concurrent Achilles’ tenotomy (Burn/tenotomy; BT) resulting in fully formed HO at the Achilles’ injury site nine weeks following injury. C57B6 and Prox1-eGFP mice labeling lymphatic endothelial cells were injured. To visualize lymphatic endothelium prior to injury, uninjured hindlimbs were harvested and processed utilizing the PEGASOS method for tissue clearing. Hindlimbs were harvested prior to injury, one, three, and nine weeks post-injury for histology. Tissue was also harvested from uninjured mice, one, and six weeks post-injury for single-cell RNA-sequencing (scRNA). Separately, we performed the BT injury in chylous ascites (Chy) mice characterized by dysfunctional lymphatic system due to an inactivating mutation in Vegfr3, a cell-surface receptor critical for the growth and survival of lymphatic endothelium. Mice were harvested nine weeks post-injury to assess the impact on HO formation. Results: Reconstructed 3-D confocal microscopy images of cleared uninjured whole-mount hindlimb tissue stained for lymphatic endothelial cell markers CD31 and LYVE1 show lymphatic vessels at the injury site. Sections from uninjured Achilles’ tendon and one, three, and nine weeks post-injury from Prox1-eGFP mice co-stained with LYVE1 show lymphatics in tendon peritenon, however, no lymphatic vessels within the tendon prior to injury. Following injury, lymphatics endothelium is disrupted, characterized by no positive Prox1-eGFP staining following injury. Interestingly, Prox1-eGFP+ cells are found within the Achilles’ tendon and Prox1-eGFP/LYVE1 double-positive cells forming a lumen are found nine weeks post-injury within the Achilles’ tendon (B; n=3/group, p*<0.5). ScRNA shows expression of lymphatic endothelial growth factor Vegfc following injury by mesenchymal progenitor cells (MPCs), while receptors for the ligand are expressed by the lymphatic endothelial cell cluster. VEGFR3 mutation leads to increased variability of HO formation following injury (E; n=4/6/group). Conclusion: These findings highlight the disruption and regeneration of lymphatic vessels following extremity trauma. For the first time, we propose a mechanism for MPC-lymphatics interaction that drives lymphatic regeneration following extremity injury through VEGFC-VEGFR3 driven lymphangiogenesis.

Divergence of Tbx4 hindlimb enhancer HLEA underlies the hindlimb loss during cetacean evolution.

The cetacean hindlimb skeleton massively decreased to only vestigial limb elements as cetaceans evolved from land to aquatic lifestyles; however, the molecular mechanism underlying this major morphological transition remains unclear. In this study, four deletions and specific substitutions were detected in cetacean hindlimb enhancer A (HLEA), an enhancer that can regulate Tbx4 expression in hindlimb tissues to control hindlimb development. Transcriptional activation of HLEA was significantly weaker in bottlenose dolphin than mice, and this was found to be closely associated with cetacean-specific deletions. Furthermore, deletions in cetacean HLEA might disrupt HOX and PITX1 binding sites, which are required for enhancer activation. The ancestral state of these deletions was investigated, and all four specific deletions were found to have occurred after the species diverged from their common ancestor, suggesting that the deletion occurred recently, during a secondary aquatic adaptation. Taking these findings together, we suggest that cetacean-specific sequence changes reduced the Tbx4 gene expression pattern, and consequently drove the gradual loss of hindlimb in cetaceans.

Hindlimb tissue composition shifts between the fetal and juvenile stages in the lamb

Hindlimb tissue composition shifts between the fetal and juvenile stages in the lamb

Abstract 11323: Role of Mir-6236 on Skeletal Muscle Angiogenesis

Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is characterized by impaired blood flow to the lower extremities. There are no medical treatments available and in severe cases, PAD may lead to critical limb ischemia requiring amputation. The pathophysiology of PAD is characterized by marked changes in gene expression and reduced angiogenesis. Although many studies have investigated the molecular mechanisms involved in PAD, less is known about the role of microRNAs (miRNA) in the pathogenesis of the disease. MiRNAs are important regulators of gene expression that bind to specific target mRNA and repress their expression. Using a mouse model of PAD, we investigated miRNAs induced by ischemia using RNAseq analysis of ischemic and non-ischemic hind limb tissue. Our data showed 77 miRNAs were upregulated and 40 miRNAs were downregulated in ischemic mouse hind limbs versus non-ischemic controls. Among these miRNAs, miR-6236 was significantly upregulated suggesting a possible role in post ischemic adaptation. We validated miR-6236 expression by qPCR (Ischemic 2.04±0.39 vs Non-Ischemic 1.0±0.11; n=5-6; p&lt;0.05). In vitro, using primary mouse microvascular endothelial cells (MVECs), ischemia induced miR-6236 expression in a time dependent manner up to 72 hrs. At the same time, ischemia caused decrease in proliferation (Ischemia (Isch) 0.12±0.03 vs Normoxia (Norm) 0.23±0.02; n=4-6; p&lt;0.05), migration (Isch 46.7±0.52 vs Norm 72.02±2.74; n=6; p&lt;0.05) and tube formation (Isch 31.5±3.58 vs Norm 82.12±6.5; n=8; p&lt;0.05), and increased apoptosis (Isch 0.94±0.05 vs Norm 0.59±0.02; n=4-5; p&lt;0.05). Treatment of mouse MVECs with miR-6236 inhibitor (miRINH, 100 nM) prior to ischemia decreased miR-6236 expression by 50% and improved proliferation (miRINH + Isch 0.21±0.01 vs Isch 0.12±0.03; n= 4; p&lt;0.05), migration (miRINH + Isch 63.71±2.08 vs Isch 46.7±0.52; n=6; p&lt;0.05), tube formation (miRINH + Isch 50±5.92 vs Isch 31.5±3.58; n=8; p&lt;0.05) and decreased ischemia-induced apoptosis (miRINH + Isch 0.64±0.04 vs Isch 0.94±0.05; n=4-5; p&lt;0.05). We conclude that miR-6236 serves as a critical regulator of endothelial cell function in ischemic skeletal muscle and can be a promising target for therapeutic skeletal muscle angiogenesis.

Plicosepalus acacia Extract and Its Major Constituents, Methyl Gallate and Quercetin, Potentiate Therapeutic Angiogenesis in Diabetic Hind Limb Ischemia: HPTLC Quantification and LC-MS/MS Metabolic Profiling.

Plicosepalus acacia (Fam. Loranthaceae) has been reported to possess hypoglycemic, antioxidant, antimicrobial, and anti-inflammatory effects. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis revealed the presence of a high content of polyphenolic compounds that are attributed to the therapeutic effects of the crude extract. In addition, methyl gallate and quercetin were detected as major phytomedicinal agents at concentrations of 1.7% and 0.062 g%, respectively, using high-performance thin layer chromatography (HPTLC). The present study investigated the effect of the P. acacia extract and its isolated compounds, methyl gallate and quercetin, on hind limb ischemia induced in type 1 diabetic rats. Histopathological examination revealed that treatment with P. acacia extract, methyl gallate, and quercetin decreased degenerative changes and inflammation in the ischemic muscle. Further biochemical assessment of the hind limb tissue showed decreased oxidative stress, increased levels of nitric oxide and endothelial nitric oxide synthase (eNOS), and enhancement of the levels of heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) in the groups treated with methyl gallate and quercetin. Expression levels of hypoxia inducible factor-1 alpha (HIF-1α), VEGF, fibroblast growth factor-2 (FGF-2), and miR-146a were upregulated in the muscle tissue of methyl gallate- and quercetin-treated groups along with downregulation of nuclear factor kappa B (NF-κB). In conclusion, P. acacia extract and its isolated compounds, methyl gallate and quercetin, mediated therapeutic angiogenesis in diabetic hind limb ischemia.

O6 Analysis of angiogenic gene profiling after E-selectin + stem cell therapy in murine ischemic limb

Abstract Introduction There remains a paucity of novel therapeutics for limb salvage in patients with critical limb ischemia (CLI) for whom revascularization procedures have failed and amputation is imminent. We have shown that E-selectin+/Mesenchymal Stem Cell (MSC) injections into the ischemic limb tissue of a CLI mouse model improves revascularization and limb function. Thus, we sought to determine a mechanism of action for E-selectin+/MSC’s pro-angiogenic and tissue salvage properties. Methods MSC were extracted from donor mice bone marrow and subsequently engineered via viral transduction with E-selectin-ires-GFP/AAV and GFP/AAV (control) to create E-selectin-GFP+/MSC vs GFP+/MSC. Intramuscular injections of E-selectin-GFP+/MSC, GFP+/MSC, or PBS were performed in a mouse model of hindlimb ischemia. Laser doppler imaging (LDI), confocal laser microscopy, and treadmill exhaustion test were utilized to determine neovascularization and limb function. RNA extraction from engineered MSC (E-selectin-GFP+/MSC vs GFP+/MSC) and ischemic hindlimb tissues treated with E-selectin-GFP+/MSC vs GFP+/MSC was performed, followed by RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis. GFP+/MSC treated hindlimb tissue served as control. Student’s t-test or ANOVA was utilized to compare means and significance set at P &amp;lt; 0.05. Results Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg LDI reperfusion (54% vs. 39% vs. 22%, P &amp;lt; 0.001), treadmill distance traversed (162 m vs. 111 m vs. 110 m, P &amp;lt; 0.01) and ischemic mouse footpad vessel density (23% vs. 14% vs. 14%, P &amp;lt; 0.01). RT2 Profiler PCR Array demonstrated pro-angiogenic gene upregulation occurred in 7 genes (Csf3, Cxcl2, Cxcl5, Serpine1, F2, Lep, Tbx1, Table I.) in E-selectin-GFP+/MSC treated ischemic leg tissue while tumour necrosis factor (TNF) was found to be downregulated, when compared with GFP+/MSC treated tissues. Of these 7 upregulated genes, CXCl2, F2, Leptin and T-box1 (Table I.) are likely produced by E-selectin-GFP+/MSC, as analysis of cellular gene expression profiles of E-selectin-GFP+/MSC also revealed upregulation by 2-fold or more in these factors when compared to GFP+/MSC. Validation of gene functions in-vivo are under investigation. Conclusion Stem cell therapy using E-selectin-GFP+/MSC, in a murine model of CLI, confers both augmented postnatal neovascularization and increased limb function. The pro-angiogenic and pro-repair effects are likely mediated by upregulation of a panel of chemokines/cytokines and down-regulation of TNF in ischemic tissues treated with E-selectin-GFP+/MSC.

Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia.

ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

Evaluation of the functionality of EUROP carcass classification system in thin-tailed and fat-tailed lambs

Aim of study was to investigate the relationship of EUROP carcass fatness and conformation classes with carcass and meat quality characteristics in thin-tailed (Kivircik) and a fat-tailed (Kangal Akkaraman) lambs. A total of 96 lamb carcasses (48 Kivircik and 48 Kangal Akkaraman breed) were examined. Distribution of carcasses in conformation and fatness classes were more limited in Kangal Akkaraman carcasses. More accurate predictions were obtained in Kivircik lambs compared to Kangal Akkaraman lambs in terms of backfat thickness, kidney knob and channel fat percentage, longissimus thoracis muscle area, weights of high-value carcass joints, weights of tissues in the hind limb and muscle/bone ratio. EUROP classification system was not appropriate for predicting meat quality in thin-tailed and fat-tailed lambs. The combination of EUROP fatness and conformation classes with cold carcass weight can be used to predict weights of high-value carcass joints and weights of hind limb tissues.

Title: Gene transfer by pyro-drive jet injector is a novel therapeutic approach for muscle diseases.

The angiogenic gene therapy is an attractive approach for the treatment of ischemic muscle diseases, including peripheral arterial disease and ischemic heart diseases. Although a variety of gene transfer methods have been developed, the efficiency of gene transfer is still limited. We have been developing the needleless high-energy bioinjector device, Pyro-drive Jet Injector (PJI), based on pyrotechnics using a combination of ignition powder and gunpowder, however, the utility of PJI in gene transfer into muscle tissues remains unclear. pcDNA3.1 plasmid containing Flag was injected to the thigh muscles of C57BL/6J mice using PJI or needle, as a control. Histological analysis demonstrated that the protein expression of Flag was observed in a wider range in PJI group than in needle group. To assess the validity of PJI for gene therapy, pcDNA3.1-human fibroblast growth factor 2 (FGF2), which has angiogenic activity and tissue protective properties, was injected into the ischemic thigh muscles with PJI or needle. ELISA assay revealed that the protein expression of FGF2 was increased in the thigh muscle tissues by PJI-mediated gene delivery. Significantly, histological analyses revealed that muscle fiber cross-sectional area and the number of endothelial marker CD31 (+) cells was increased in ischemic hind-limb tissues of the PJI-FGF2 group but not in those of needle-FGF2 group. To expand the applicability of the PJI-mediated gene transfer, pcDNA3.1-venus plasmid was injected into murine hearts with PJI or needle. PJI method was successful in gene transfer into murine hearts, especially into cardiomyocytes, with high efficiency when compared to needle method. Collectively, the non-needle, non-liposomal and non-viral gene transfer by PJI could be a novel therapeutic approach for muscle diseases.

Pitx genes in development and disease.

Homeobox genes encode sequence-specific transcription factors (SSTFs) that recognize specific DNA sequences and regulate organogenesis in all eukaryotes. They are essential in specifying spatial and temporal cell identity and as a result, their mutations often cause severe developmental defects. Pitx genes belong to the PRD class of the highly evolutionary conserved homeobox genes in all animals. Vertebrates possess three Pitx paralogs, Pitx1, Pitx2, and Pitx3 while non-vertebrates have only one Pitx gene. The ancient role of regulating left-right (LR) asymmetry is conserved while new functions emerge to afford more complex body plan and functionalities. In mouse, Pitx1 regulates hindlimb tissue patterning and pituitary development. Pitx2 is essential for the development of the oral cavity and abdominal wall while regulates the formation and symmetry of other organs including pituitary, heart, gut, lung among others by controlling growth control genes upon activation of the Wnt/ß-catenin signaling pathway. Pitx3 is essential for lens development and migration and survival of the dopaminergic neurons of the substantia nigra. Pitx gene mutations are linked to various congenital defects and cancers in humans. Pitx gene family has the potential to offer a new approach in regenerative medicine and aid in identifying new drug targets.

Ultrasound-Mediated Microbubble Cavitation Transiently Reverses Acute Hindlimb Tissue Ischemia through Augmentation of Microcirculation Perfusion via the eNOS/NO Pathway

Ultrasound-mediated microbubble cavitation improves perfusion in chronic limb and myocardial ischemia. The purpose of this study was to determine the effects of ultrasound-mediated microbubble cavitation in acute limb ischemia and investigate the mechanism of action. The animal with acute hindlimb ischemia was established using male Sprague-Dawley rats. The rats were randomly divided into three groups: intermittent high-mechanical-index ultrasound pulses combined with microbubbles (ultrasound [US] + MB group), US alone (US group) and MB alone (MB group). Both hindlimbs were treated for 10 min. Contrast ultrasound perfusion imaging of both hindlimbs was performed immediately and 5, 10, 15, 20 and 25 min after treatment. The role of the nitric oxide (NO) pathway in increasing blood flow in acutely ischemic tissue was evaluated by inhibiting endothelial nitric oxide synthase (eNOS) with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). In the US + MB group, microvascular blood volume and microvascular blood flow of the ischemic hindlimb were significantly increased after treatment (both p values <0.05), while the microvascular flux rate (β) increased, but not significantly (p > 0.05). The increases were observed immediately after treatment, and had dissipated by 25 min. Changes in the US and MB groups were minimal. Inhibitory studies indicated cavitation increased phospho-eNOS concentration in ischemic hindlimb muscle tissue, and the increase was significantly inhibited by L-NAME (p < 0.05). Ultrasound-mediated microbubble cavitation transiently increases local perfusion in acutely ischemic tissue, mainly by improving microcirculatory perfusion. The eNOS/NO signaling pathway appears to be an important mediator of the effect.

Abstract 4706: Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis

Abstract Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death among men in America. Aggressive prostate tumors that rapidly metastasize to other organs such as bones account for high morbidity and mortality. Metastatic PCa is resistant to androgen depletion therapy, and increased expression of androgen receptor (AR) coregulator Steroid Receptor Coregulator-2 (SRC-2) remains a major underlying driver of lethal PCa progression. Increased expression of SRC-2 positively correlates with PCa recurrence and poor disease outcome. SRC-2 directly binds to AR and regulates its transcriptional activity even in castrate-levels of androgen. Although the role of SRC-2 as a coactivator of AR is well established, we recently discovered that SRC-2 can also function as a co-repressor of AR on certain gene promoters such as mitochondrial deacetylase Sirtuin 3 (SIRT3). Analysis of human PCa databases revealed that expression of SIRT3 is reduced in prostate tumors as the disease progresses to metastatic PCa, and there exists an inverse correlation with SRC-2 expression. To confirm that SRC-2 is functioning as a co-repressor on SIRT3 promoter we performed chromatin immunoprecipitation (ChIP) and identified strong occupancy of SRC-2 on the SIRT3 promoter along with AR, whereas SRC-2 knockdown reduced AR enrichment. Consistently with this observation SRC-2 or AR knockdown significantly increased SIRT3 expression in PCa lines C4-2 and 22RV1, validating that SRC-2 functions as a transcriptional co-repressor of AR on SIRT3 promoter. This suggests that AR/SRC-2 can transcriptionally silence potential tumor suppressors. Next, we investigated the role of SIRT3 in PCa, and adenovirus-mediated ectopic expression of SIRT3 significantly impaired cell cycle progression arresting TRAMP-C2 and C4-2 cells in G0/G1 phase with reduced cell growth and viability, suggesting SIRT3 may function as a tumor suppressor in PCa. Since SRC-2 represses SIRT3 gene expression we analyzed mRNA expression of SRC-2 and SIRT3 in metastatic lesions from human prostate cancer patients using an advanced prostate cancer genomics dataset. We found that increased levels of SRC-2 with a concomitant reduction in SIRT3 expression is prevalent in metastatic lesions of PCa patients that spread to bones and lymph nodes. We used intra-iliac artery (IIA) injection to selectively deliver C4-2 cells expressing stable knockdown of SRC-2 (shSRC-2) or NTshRNA into the hind limb tissues. After 12 weeks we found SRC-2 ablation almost completely abolished PCa colonization in the bone whereas all animals transplanted with C4-2-NTshRNA developed bone metastasis. In conclusion, our study indicates that transcriptional repression of tumor suppressor SIRT3 by SRC2 may function as an oncogenic driver of lethal PCa progression to the bone. This work is supported by DoD grant: W81XWH-16-1-0297, NCI grant: K22CA207578 and Roswell Park Alliance Foundation. Citation Format: Mayrel Palestino Dominguez, Abhisha Sawant Dessai, Eriko Katsuta, Tao Dai, Subhamoy Dasgupta. Transcriptional repression of mitochondrial deacetylase SIRT3 promotes prostate cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4706.

Effects of edoxaban and warfarin on vascular remodeling: Atherosclerotic plaque progression and collateral artery growth

Background and purposeOral anticoagulation prevents thromboembolism in atrial fibrillation. Factor Xa inhibitors, like edoxaban, are known to reduce inflammation and proliferation of smooth muscle cells, while vitamin K antagonism can cause vascular calcific damage. The influence of edoxaban compared to warfarin on vascular remodeling, atherosclerosis and arteriogenesis is unknown. Experimental approachApolipoprotein E knockout (ApoE −/−) mice were fed cholesterol-rich diet alone (control, co), with warfarin+vitamin K1 (warf) or with edoxaban (Edo) for 8 weeks. After 6 weeks, femoral artery ligation was performed. Key resultsThere was no difference in hind-limb perfusion restoration between the three groups after 14 days (Co 0.36 ± 0.05 vs. Warf 0.39 ± 0.09 (p = .39), Co vs. Edo 0.51 ± 0.06 (p = .089), Warf vs. Edo (p = .83)) after ligation. Immuno-histologically, there was no difference in smooth muscle cell count in both hindlimbs between the three groups or in the amount of perivascular macrophages in collateral-bearing hindlimb tissue. Edoxaban showed the lowest amount of plaque tissue in the aortic sinus tissue (Co 74 ± 11% vs. Edo 62 ± 12% (p = .024), Co vs. Warf 69 ± 14% (p = .30), Edo vs. Warf (p = .14)) as well as the least amount of fibrosis (Co 3.1 ± 0.9% vs. Edo 1.7 ± 0.6% (p = .027), Co vs. Warf 4.1 ± 0.7% (p = .081), Edo vs. Warf (p < .001)). No difference in mRNA content of inflammatory cytokines in muscle tissue or spleen was detected between the three groups. Conclusion and implicationsThese data suggest that treatment with edoxaban unlike warfarin prevents vascular maladaptive remodeling, which may be clinically important.

Novel Contribution of Myeloperoxidase‐derived Hypochlorous Acid to 20‐HETE Production Post‐Ischemia

Ischemia‐induced angiogenesis occurs as a mechanism to minimize the tissue damage caused by atherosclerotic diseases but is often insufficient to maintain adequate tissue perfusion and results in critical limb ischemia and amputation. The discovery of novel factors that increase angiogenesis in response to ischemia is therefore of clinical relevance.Recently, we discovered that 20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by a cytochrome P450 (CYP) 4A/F ω‐hydroxylases, is a novel contributor of ischemia‐induced angiogenesis based on the following two major findings: 1) pharmacological interference of the CYP4A/20‐HETE axis attenuates ischemic‐induced angiogenesis; and 2) the expression of cyp4a12a and its product, 20‐HETE are both elevated in ischemic tissues of mice models. The resulting increase in 20‐HETE is endothelial in origin. However, the factors that cause endothelial cells (EC) to produce 20‐HETE following ischemia are largely unknown. The aim of these studies was to investigate the mechanism of post‐ischemic endothelial 20‐HETE increases. Inflammatory cells such as macrophages and neutrophils are the first responders to ischemia injury. Thus, we hypothesized that inflammatory cells contribute to post‐ischemic endothelial increases of 20‐HETE.We first examined the potential contribution of inflammatory response to hindlimb ischemia‐induced angiogenesis using immune‐deficient SCID mice. Following a femoral artery ligation and comparing SCID mice to Balb/C control mice, SCID mice exhibited impaired blood perfusion (55 ± 9% vs 89 ± 10%), decreased micro‐vessel counts (capillary:fiber ratio of 1.5 ± .25 vs 2.5 ± 3.75), as well as an inability to produce 20‐HETE (7.5 ± 2.5 vs 29 ± 5 pg/mg of protein).Furthermore, immune‐competent Balb/c mice depleted of macrophages or neutrophils showed that ischemia failed to induce 20‐HETE production in Balb/c depleted of neutrophils (13 ± 1.5 vs 35 ± 5 pg/mg of protein), accompanied with compromised blood perfusion (58 ± 10% vs 82 ± 9%), but not those depleted of macrophages. Neutrophils are known to release Myeloperoxidase (MPO) at sites of inflammation. The possibility that MPO contributes to ischemia‐induced 20‐HETE production was investigated using MPO−/− mice. Remarkably, the ischemic hind limb tissues of MPO−/− mice produce negligible amount of 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein). MPO is known to produce hypochlorous acid (HOCl). Therefore, we further studied the role of MPO‐derived HOCl in 20‐HETE production in cultured human EC. The addition of physiological amounts of either MPO or HOCl markedly stimulated the expression of 20‐HETE synthase CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein); potentially via a HIF1‐a dependent mechanism.These observations suggest for the first time that neutrophil‐derived MPO critically contributes to the 20‐HETE increases post‐ischemia via a mechanism involving the generation of HOCl and activation of the HIF pathway in endothelium.Support or Funding InformationAHA [11SDG6870004(AMG), 17GRNT33430003(AMG)]NIH [HL34300(MLS), DK38226(JRF)]RA Welch Foundation [GL625910(JRF)]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tumor suppressor protein p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis

ObjectiveThe tumor suppressor protein p53 regulates angiogenesis and is a key regulatory mediator of cellular apoptosis, proliferation, and growth. p53 expression is induced in response to ischemia; however, its role in regulating ischemia-induced angiogenesis and arteriogenesis remains undefined. The objective of this study was to define the role of p53 in regulating ischemia-induced angiogenesis and arteriogenesis and to identify mechanisms by which this regulation occurs in vivo. MethodsSurgically induced hindlimb ischemia or mesenteric artery ligation was performed in wild-type (p53+/+) and p53 knockout (p53−/−) mice. Limb perfusion and revascularization were assessed by laser Doppler perfusion imaging, capillary density, and collateral artery development. Mesenteric collateral artery flow and development were determined by arterial flow measurement and by histologic analysis, respectively. An in vitro aortic ring assay was performed on p53+/+ and p53−/− aortic tissue to evaluate endothelial function. The p53 inhibitor and activator pifithrin-α and quinacrine, respectively, were used to modulate p53 activity in vivo after ischemia. ResultsAbsence of p53 in mice resulted in increased limb perfusion (P < .05), capillary density (P < .05), and collateral artery development (P < .05) after induction of hindlimb ischemia. In the nonischemic mesenteric artery ligation model of arteriogenesis, p53 expression was induced in collateral arteries and increased arterial blood flow in mice lacking p53 (P < .05). Lack of p53 decreased apoptosis in ischemic hindlimb tissue (P < .05) and increased proangiogenic factors hypoxia-inducible factor 1α and vascular endothelial growth factor (VEGF). Endothelial cell outgrowth in vitro increased in the absence of p53 (P < .05). Pharmacologic augmentation of p53 expression after ischemia impaired perfusion and collateral artery formation and decreased VEGF levels (P < .05). Conversely, inhibition of p53 with pifithrin-α augmented limb perfusion (P < .05) and collateral artery formation (P < .05) and increased protein levels of hypoxia-inducible factor 1α and VEGF. Pharmacologic augmentation and inhibition of p53 had no significant effect in mice lacking p53. Conclusionsp53 negatively regulates ischemia-induced angiogenesis and arteriogenesis. Inhibition of p53 increases ischemia-induced arteriogenesis and limb perfusion and thus represents a potential therapeutic strategy for arterial occlusive disease.