Mouse Model Of Hindlimb Ischemia Research Articles

CML-074 Pathogenesis of Tyrosine Kinase Inhibitor–Associated Vascular Toxicity in Chronic Myeloid Leukemia

Evidence has emerged demonstrating an association between treatment with certain tyrosine kinase inhibitors (TKIs) and vascular adverse events (VAEs) in chronic myeloid leukemia (CML). An increase in VAEs has been documented with ponatinib, nilotinib, and dasatinib but not with imatinib and bosutinib. We used an endothelial cell (HUVEC) model and an in-vivo hindlimb ischemia mouse model to study TKI-associated VAEs. We show that compared with control-treated cells, ponatinib increased HUVEC apoptosis by 97% and inhibited their VEGF-induced migration by 35%. HUVEC tube formation was significantly abrogated by ponatinib as evidenced by an 87% reduction in tube number, 68% reduction in segment length, 50% reduction in junction number, and 55% increase in the occurrence of isolated segments. Ponatinib's suppressive effect on tube formation was partially rescued by VEGFR2 overexpression, a known ponatinib off-target. Dasatinib also dramatically disrupted tube formation as evidenced by a 97% reduction in tube number, 95% reduction in segment length, 88% reduction in junction number, and 300% increase in the occurrence of isolated segments. Nilotinib was associated with reduced cell viability, whereas imatinib and bosutinib did not affect these processes. Furthermore, HUVECs treated with ponatinib, dasatinib, and nilotinib revealed a differential angiogenesis-related gene expression pattern. The expression profile of imatinib- and bosutinib-treated cells was similar to that of control-treated cells. In our in-vivo model, unilateral femoral artery excision was performed to induce ischemia and stimulate new blood vessel formation in TKI-treated mice. Imatinib-treated mice showed a similar recovery pattern to that of control mice as assessed via Doppler imager. Bosutinib- and dasatinib-treated mice showed an initial delayed recovery but reached an overall recovery of 85% within 28 days. In contrast, ponatinib-treated mice reached overall perfusion of 60% around day 7 without further improvement. These results are in line with the clinical VAE profiles of ponatinib, dasatinib, and nilotinib, implicating endothelial toxicity related to these TKIs and supporting a direct effect of these TKIs on vascular cells. However, the in-vivo effect of these TKIs on endothelial pathogenesis requires further investigation. Understanding TKI-induced VAE pathogenesis could contribute to the future development of safer next-generation TKIs and could potentially influence TKI-personalized tailoring for CML patients.

Increasing angiogenic efficacy of conditioned medium using light stimulation of human adipose-derived stem cells

Conditioned medium (CM) contains various therapeutic molecules produced by cells. However, the low concentration of therapeutic molecules in CM is a major challenge for successful tissue regeneration. Here, we aim to develop a CM enriched in angiogenic paracrine factors for the treatment of ischemic diseases. Combining spheroidal culture and light irradiation significantly upregulates the angiogenic factor expression in human adipose-derived stem cells (hADSCs). Spheroids of light-irradiated hADSCs (SR group) show significantly enhanced expression of angiogenic paracrine factors compared with spheroids without light stimulation. Enhanced viability, migration, and angiogenesis are observed in cells treated with CM derived from the SR group. Furthermore, we performed in vivo experiments using a mouse hindlimb ischemia model; the results demonstrate that CM derived from densely cultured spheroids of light-irradiated hADSCs induced increased angiogenesis in vivo. In conclusion, our proposed approach of using light to stimulate stem cells may overcome the major drawbacks of CM-based therapies.

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers

BackgroundWhile rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers.MethodsThe angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial.ResultsHypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed.ConclusionsThe present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy.Trial registration: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784

Salvianolic Acid B Alleviates Limb Ischemia in Mice via Promoting SIRT1/PI3K/AKT Pathway-Mediated M2 Macrophage Polarization.

Salvianolic acid B (Sal B) is an effective treatment agent for ischemic disease in China. However, Sal B's effects on peripheral arterial disease (PAD) and its mechanism remains poorly understood. Macrophage polarization plays a crucial role in PAD. Nevertheless, treatment modalities that increase the population of anti-inflammatory (M2) macrophages are limited. This study aimed to explore the protective effects of Sal B on limb perfusion and investigate the mechanism of Sal B-induced macrophage polarization. C57BL/6 male mice (6 weeks) were randomized into control, Model + NS, and Model + Sal B groups (n = 5). Then, we established a hind limb ischemia mouse model to assess the Sal B's role (15 mg/kg/d) in PAD. We quantified the blood perfusion via laser speckle contrast imaging (LSCI) and measured the capillary density and muscle edema with CD31 and H&E staining. The Sal B-induced macrophage polarization was confirmed by qPCR and ELISA. The results showed that the Sal B group exhibited a significant improvement in the blood perfusion, capillary density, muscle edema, and M2 markers gene expressions. Cell migration and tube formation were promoted in the endothelial cells stimulated with a culture supernatant from Sal B-treated macrophages. In contrast, endothelial functions improved by Sal B-treated macrophages were impaired in groups treated with SIRT1 and PI3K inhibitors. These findings provide evidence for Sal B's protective role in PAD and demonstrate the enhancement of macrophage polarization via the SIRT1/PI3K/AKT pathway.

Abstract 548: Prolonging The Viability Of Induced Pluripotent Stem Cell-derived Endothelial Cells For Treatment Of Peripheral Arterial Disease Using Engineered Biomaterials

Dysfunctional endothelial cells (EC) can restrict flow in the extremities, leading to limb ischemia. We aim to improving revascularization of ischemic limbs using cell therapy, augmented with spatially patterned scaffolds that promote survival. The purpose was to evaluate the effect spatially patterned scaffolds on endothelial cell survival. The biophysical parameters of collagen scaffolds (alignment, crosslinking, and fibril diameter) were characterized with atomic force microscopy and degradation assays. Cytoskeletal arrangement of primary human coronary artery ECs and human induced pluripotent stem cell (iPSC)-derived ECs on collagen scaffolds was evaluated by F-actin staining. To evaluate the effect of mechanical properties on cell viability in vitro, Live/Dead and MTS assays were used. Cell seeded scaffolds were implanted into a mouse hindlimb ischemia model, and cell survival was tracked by bioluminescent imaging. Both primary EC and iPSC-ECs reorganized their F-actin cytoskeleton corresponding to the alignment of the fibrils, irrespective of crosslinking degree, stiffness or fibril diameter. When cells were grown in hypoxic mimicking ischemia however, viability was improved using aligned scaffolds with 200 nm fibril diameter. Degradation assays showed that scaffolds with higher crosslinking had significantly more durability. When cell-seeded scaffolds were implanted to the ischemic limb, the aligned scaffolds with 200nm fibril diameter had the highest survival, confirming in vitro studies. Aligned collagen scaffolds with 200nm fibril diameter and high cross-linking degree led to improved viability of primary and iPSC-ECs in a mouse model of PAD.

Peripheral Serum Exosomes Isolated from Patients with Acute Myocardial Infarction Promote Endothelial Cell Angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α Pathway.

PurposeAngiogenesis is required for improving myocardial function and is a key factor in long-term prognosis after an acute myocardial infarction (AMI). Although exosomes are known to play a crucial role in angiogenesis, the role of peripheral exosomes in angiogenic signal transduction in patients with AMI remains unclear. Here, we explored the effect of exosomes extracted from the peripheral serum of AMI patients on angiogenesis and elucidated the downstream pathways.Patients and MethodsSerum exosomes were obtained from patients with AMI (AMI-Exo) and healthy individuals (Con-Exo). The exosomes were cocultured with human umbilical vein endothelial cells (HUVECs) in vitro, with aortic rings ex vivo, and were used to treat mouse hind-limb ischemia and mouse AMI model in vivo.ResultsAMI-Exo raised HUVEC proliferation, tube formation, and migration, and enhanced microvessel sprouting from aortic rings compared to Con-Exo, both in vitro and ex vivo. Quantitative reverse transcription-polymerase chain reaction revealed that the abundance of miR-126-3p, a crucial regulator of angiogenesis, was increased in AMI-Exo. The inhibition of miR-126-3p decreased the benefits of AMI-Exo treatment, and miR-126-3p upregulation enhanced the benefits of Con-Exo treatment in HUVECs, aortic rings, the mouse hind-limb ischemia model, and the mouse AMI model. Knockdown and overexpression analyses revealed that miR-126-3p regulated angiogenesis in HUVECs by directly targeting tuberous sclerosis complex 1 (TSC1). Moreover, we found that miR-126-3p could inhibit TSC1 expression, which further activated mTORC1 signaling and increased HIF-1α and VEGFA expression, ultimately promoting angiogenesis.ConclusionCollectively, our results provide a novel understanding of the function of exosomes in angiogenesis post AMI. We demonstrated that exosomes from the peripheral serum of AMI patients promote angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α signaling pathway.

The transplantation of rapamycin-treated senescent human mesenchymal stem cells with enhanced proangiogenic activity promotes neovascularization and ischemic limb salvage in mice.

Few therapies can reverse the proangiogenic activity of senescent mesenchymal stromal/stem cells (MSCs). In this study, we investigated the effects of rapamycin on the proangiogenic ability of senescent human umbilical cord MSCs (UCMSCs). An in vitro replicative senescent cell model was established in cultured UCMSCs. We found that late passage (P25 or later) UCMSCs (LP-UCMSCs) exhibited impaired proangiogenic abilities. Treatment of P25 UCMSCs with rapamycin (900 nM) reversed the senescent phenotype and notably enhanced the proangiogenic activity of senescent UCMSCs. In a nude mouse model of hindlimb ischemia, intramuscular injection of rapamycin-treated P25 UCMSCs into the ischemic limb significantly promoted neovascularization and ischemic limb salvage. We further analyzed the changes in the expression of angiogenesis-associated genes in rapamycin-primed MSCs and found higher expression of several genes related to angiogenesis, such as VEGFR2 and CTGF/CCN2, in primed cells than in unprimed MSCs. Taken together, our data demonstrate that rapamycin is a potential drug to restore the proangiogenic activity of senescent MSCs, which is of importance in treating ischemic diseases and tissue engineering.

Dual Function of a in vivo Albumin-Labeling Tracer for Assessment of Blood Perfusion and Vascular Permeability in Peripheral Arterial Disease by PET.

BackgroundPeripheral arterial disease (PAD) leads to tissue ischemia in the extremities. Enhanced vascular permeability plays a critical role in targeted delivery of drugs for effective therapeutic angiogenesis and resultant blood perfusion recovery. However, optimal tracers for evaluating this process in PAD patients are lacking. At this time, we employed a novel in vivo albumin-labeling tracer of dual function, termed as 18F-NEB, to assess blood perfusion as well as vascular permeability by positron emission tomography (PET).Methods and ResultsAfter successful establishment of mouse hindlimb ischemia (HI) model, static PET imaging was performed 15 min and 2 h post injection (p.i.) of 18F-NEB at 1, 3, 5, 7, 10 and 14 days post-surgery respectively. Gradual recovery of blood supply was detected by PET scan 15 min p.i. and collaborated by serial Laser Doppler. In addition, the highest vascular permeability observed by high local uptake of 18F-NEB at 2 h p.i. was consistent with histological examinations. Furthermore, we quantitatively evaluated the effect of vascular endothelial growth factor (VEGF) stimulus on vascular permeability and blood perfusion by PET scan using 18F-NEB probe in HI model, which were also confirmed by immunohistological results.ConclusionThe application of 18F-NEB probe alone by PET can successfully achieve dual imaging of blood perfusion as well as vascular permeability at different time points p.i. and monitor their responses to therapy in PAD model. The simple labeling approach and multipurpose feature suggest the great promise of using this imaging probe in theranostic applications for treating ischemic disease.

Hypoxia pretreatment improves the therapeutic potential of bone marrow mesenchymal stem cells in hindlimb ischemia via upregulation of NRG-1.

Mesenchymal stem cells (MSCs) are considered a promising treatment for ischemic diseases, but their use is limited due to poor survival after injection. Hypoxia can significantly enhance the survival of MSCs. This study aimed to investigate hypoxia pretreatment of bone marrow mesenchymal stem cells (BM-MSCs) in hindlimb ischemia (HI) and the underlying mechanism. The HI mouse model was established and human BM-MSCs were injected into ischemic skeletal muscles. The blood flow reperfusion and capillary density were measured. In vitro, human BM-MSC cells were treated with hypoxia. The expression of NRG-1 and associated angiogenic factors were measured after knockdown or overexpression of NRG-1. The conditioned medium (CdM) of BM-MSCs was prepared and co-cultured with human umbilical vein endothelial cells (HUVECs), and then, the proliferation, migration, and angiogenesis of HUVECs were detected. After hypoxia pretreatment, NRG-1 expression, clone formation, proliferation, and angiogenic factor secretion from BM-MSCs were increased, while knockdown of NRG-1 reversed these results. In normoxia condition, overexpression of NRG-1 enhanced above factors. Additionally, hypoxia pretreatment of BM-MSCs induced the proliferation and migration of HUVECs and angiogenesis. Moreover, the injection of hypoxia pretreatment of BM-MSCs improved blood reperfusion and capillary density in HI mice, while knockdown of NRG-1 reversed the effect. Furthermore, the PI3K inhibitor and activator reversed the effect of NRG-1 overexpression and knockdown on angiogenesis. We concludes thathypoxia pretreatment of BM-MSCs facilitates angiogenesis and alleviates HI injury via NRG-1/PI3K/AKT pathway.

YB-1 Recruits Drosha to Promote Splicing of pri-miR-192 to Mediate the Proangiogenic Effects of H2S.

Aims: The genes targeted by miRNAs have been well studied. However, little is known about the feedback mechanisms to control the biosynthesis of miRNAs that are essential for the miRNA feedback networks in the cells. In this present study, we aimed at examining how hydrogen sulfide (H2S) promotes angiogenesis by regulating miR-192 biosynthesis. Results: H2S promoted in vitro angiogenesis and angiogenesis in Matrigel plugs embedded in mice by upregulating miR-192. Knockdown of the H2S-generating enzyme cystathionine γ-lyase (CSE) suppressed in vitro angiogenesis, and this suppression was rescued by exogenous H2S donor NaHS. Plakophilin 4 (PKP4) served as a target gene of miR-192. H2S up-regulated miR-192 via the VEGFR2/Akt pathway to promote the splicing of primary miR-192 (pri-miR-192), and it resulted in an increase in both the precursor- and mature forms of miR-192. H2S translocated YB-1 into the nuclei to recruit Drosha to bind with pri-miR-192 and promoted its splicing. NaHS treatment promoted angiogenesis in the hindlimb ischemia mouse model and the skin-wound-healing model in diabetic mice, with upregulated miR-192 and downregulated PKP4 on NaHS treatment. In human atherosclerotic plaques, miR-192 levels were positively correlated with the plasma H2S concentrations. Innovation and Conclusion: Our data reveal a role of YB-1 in recruiting Drosha to splice pri-miR-192 to mediate the proangiogenic effect of H2S. CSE/H2S/YB-1/Drosha/miR-192 is a potential therapeutic target pathway for treating diseases, including organ ischemia and diabetic complications. Antioxid. Redox Signal. 36, 760-783. The Clinical Trial Registration number is 2016-224.

MiR-548j-5p regulates angiogenesis in peripheral artery disease

Peripheral artery disease (PAD) is a vascular disease involving diffuse atherosclerosis, and is associated with increased cardiovascular mortality and morbidity. Critical limb ischemia (CLI) is the most severe complication of PAD. In addition to medical and interventional treatment, therapeutic angiogenesis is a novel therapy for PAD. Circulating microRNAs (miRNAs) are considered key regulators of gene expression, but their role in ischemic-induced angiogenesis is poorly-characterized. There is currently a limited understanding of the specific miRNAs associated with PAD. To determine the regulation of miRNAs, we obtained miRNA profiles using RNA isolated from patients with PAD and a control group. The effects of specific miRNAs on angiogenesis were evaluated by assessing the in vitro angiogenic function of endothelial progenitor cells (EPCs), performing an in vivo angiogenesis assay, and employing a mouse hindlimb ischemic model. Our results demonstrated that circulating miR-548j-5p was significantly reduced in patients with PAD as compared with the controls. miR-548j-5p promoted EPC angiogenesis by enhancing migration and tube formation. The endothelial nitric oxide synthase (NOS) and stromal cell-derived factor (SDF)-1 signaling pathways appeared to be potential targets of miR-548j-5p. Furthermore, the results of a directed in vivo angiogenesis assay of EPCs and a hindlimb ischemia mouse model demonstrated that miR-548j-5p enhanced the capillary density and blood flow recovery in hindlimb ischemia. In conclusion, our data indicated that up-regulation of miR-548j-5p promotes angiogenesis in ischemic tissue and may represent a novel therapeutic approach for PAD.

Quenching Epigenetic Drug Resistance Using Antihypoxic Microparticles in Glioblastoma Patient-Derived Chips.

Glioblastoma (GBM) is one of the most intractable tumor types due to the progressive drug resistance upon tumor mass expansion. Incremental hypoxia inside the growing tumor mass drives epigenetic drug resistance by activating nongenetic repair of antiapoptotic DNA, which could be impaired by drug treatment. Hence, rescuing intertumor hypoxia by oxygen-generating microparticles may promote susceptibility to antitumor drugs. Moreover, a tumor-on-a-chip model enables user-specified alternation of clinic-derived samples. This study utilizes patient-derived glioblastoma tissue to generate cell spheroids with size variations in a 3D microchannel network chip (GBM chip). As the spheroid size increases, epigenetic drug resistance is promoted with inward hypoxia severance, as supported by the spheroid size-proportional expression of hypoxia-inducible factor-1a in the chip. Loading antihypoxia microparticles onto the spheroid surface significantly reduces drug resistance by silencing the expression of critical epigenetic factor, resulting in significantly decreased cell invasiveness. The results are confirmed in vitro using cell line and patient samples in the chip as well as chip implantation into a hypoxic hindlimb ischemia model in mice, which is an unprecedented approach in the field.

Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair.

Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation.Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration.Results: In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs.Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.

S100A8 and S100A9 are elevated in chronically threatened ischemic limb muscle and induce ischemic mitochondrial pathology in mice

ObjectiveThe objective of the present study was to determine whether elevated levels of S100A8 and S100A9 (S100A8/A9) alarmins contribute to ischemic limb pathology. MethodsGastrocnemius muscle was collected from control patients without peripheral arterial disease (PAD; n = 14) and patients with chronic limb threatening limb ischemia (CLTI; n = 14). Mitochondrial function was assessed in permeabilized muscle fibers, and RNA and protein analyses were used to quantify the S100A8/A9 levels. Additionally, a mouse model of hindlimb ischemia with and without exogenous delivery of S100A8/A9 was used. ResultsCompared with the non-PAD control muscles, CLTI muscles displayed significant increases in the abundance of S100A8 and S100A9 at both mRNA and protein levels (P < .01). The CLTI muscles also displayed significant impairment in mitochondrial oxidative phosphorylation and increased mitochondrial hydrogen peroxide production compared with the non-PAD controls. The S100A8/A9 levels correlated significantly with the degree of muscle mitochondrial dysfunction (P < .05 for all). C57BL6J mice treated with recombinant S100A8/A9 displayed impaired perfusion recovery and muscle mitochondrial impairment compared with the placebo-treated mice after hindlimb ischemia surgery. These mitochondrial deficits observed after S100A8/A9 treatment were confirmed in the muscle cell culture system under normoxic conditions. ConclusionsThe S100A8/A9 levels were increased in CLTI limb muscle specimens compared with the non-PAD control muscle specimens, and the level of accumulation was associated with muscle mitochondrial impairment. Elevated S100A8/A9 levels in mice subjected to hindlimb ischemia impaired perfusion recovery and mitochondrial function. Together, these findings suggest that the inflammatory mediators S100A8/A9 might be directly involved in ischemic limb pathology.

Abstract 10451: A Potential Therapeutic Antibody to Induce Collateral Formation in Cardiovascular Diseases

Introduction: Lack of efficient collateralisation during vascular occlusion leads to tissue ischemia in coronary, peripheral and cerebrovascular disease. Patients with peripheral, coronary, and cerebrovascular disease have elevated levels of the anti-angiogenic isoform of VEGF (VEGF-A 165 b), which correlate with poorer outcomes. In experimental animals, VEGF-A 165 b neutralisation improves blood flow and collateralisation after femoral or coronary artery ligation. Hypothesis: We tested the hypothesis that a potent, high affinity, humanised neutralising antibody to VEGF-A 165 b could have therapeutic potential in models of cardiovascular disease. Methods: A mouse monoclonal antibody to VEGF-A 165 b was humanised by cloning and site directed mutagenesis. Multiple clones were screened, and one with high affinity (measured by biolayer interferometry) was amplified. The efficacy in vitro was tested using endothelial cell migration assays and in vivo using the hindlimb ischemia (HLI) model in C57Bl6 mice fed a high fat high sucrose (HFHS) diet, and in Zucker Diabetic Fatty rats. Results: One clone (HC4LC2) had a VEGF-A 165 b affinity of 600pM. 0.3μg/ml of this clone reversed the inhibitory effect of recombinant human VEGF-A 165 b (31±4%, N=6 of VEGF-A 165 a alone) on migration of endothelial cells to 40ng/ml rhVEGF-A 165 a (89±9% N=3). Human monocyte dependent inhibition of VEGF-A 165 a mediated migration was dose dependently reversed by HC4LC2 (EC50, 55ng/ml, N=9 per concentration). HLI in HFHS mice impaired blood flow after 28 days in IgG treated mice (70±7.7% of pre-ischemic blood flow, N=6), which was restored to normal (93.3±5.5, p&lt;0.05, N=8) by intraperitoneal injection of 1mg/kg HC4LC2. Blood flow recovery after HLI was also seen in ZDF rats treated with HC4LC2 (106±16% of pre-ischemic flow, N=5) compared with IgG treated animals (76±.9%, N=5, p&lt;0.05, two-way ANOVA, all values mean ±SEM). Conclusions: These results indicate that HC4LC2 is a humanised, high affinity neutralising antibody to VEGF-A 165 b that could have therapeutic potential for patients with cardiovascular disease, by enhancing collateral formation.

Area light source-triggered latent angiogenic molecular mechanisms intensify therapeutic efficacy of adult stem cells.

Light‐based therapy such as photobiomodulation (PBM) reportedly produces beneficial physiological effects in cells and tissues. However, most reports have focused on the immediate and instant effects of light. Considering the physiological effects of natural light exposure in living organisms, the latent reaction period after irradiation should be deliberated. In contrast to previous reports, we examined the latent reaction period after light exposure with optimized irradiating parameters and validated novel therapeutic molecular mechanisms for the first time. we demonstrated an organic light‐emitting diode (OLED)‐based PBM (OPBM) strategy that enhances the angiogenic efficacy of human adipose‐derived stem cells (hADSCs) via direct irradiation with red OLEDs of optimized wavelength, voltage, current, luminance, and duration, and investigated the underlying molecular mechanisms. Our results revealed that the angiogenic paracrine effect, viability, and adhesion of hADSCs were significantly intensified by our OPBM strategy. Following OPBM treatment, significant changes were observed in HIF‐1α expression, intracellular reactive oxygen species levels, activation of the receptor tyrosine kinase, and glycolytic pathways in hADSCs. In addition, transplantation of OLED‐irradiated hADSCs resulted in significantly enhanced limb salvage ratio in a mouse model of hindlimb ischemia. Our OPBM might serve as a new paradigm for stem cell culture systems to develop cell‐based therapies in the future.

Delivery of extracellular matrix-enriched stem cells encapsulated with enzyme-free pH-sensitive polymer for enhancing therapeutic angiogenesis

At clinical transplantation of human adipose derived stem cells (hADSCs), a proteolytic enzyme treatment for harvesting hADSCs must be used but usually causes the disruption of extracellular matrices (ECMs) interconnection. As ECMs-poor hADSCs partially receive the cellular signals, hADSCs deactivated or undergo cell death. Herein, we have proposed a method for delivering ECMs-enriched hADSCs encapsulated with a new polymer for transplanting into ischemic wound sites to enhance the therapeutic angiogenic efficacy. Enzyme free pH-sensitive polymer (EFP) was synthesized and coated on tissue culture plate for hADSCs culture. When the hADSCs were cultured on EFP-coated surfaces, they were easily detached with pH 6.0 buffer solution and dissolved EFP wrapped hADSCs with more ECMs for 48 h. When ECMs enriched-hADSCs were re-adhered, the cells secreted more angiogenic factors under serum free and hypoxic cell culture condition. When injecting ECMs-enriched hADSCs to mouse hindlimb ischemia models, the blood perfusion and blood vessel regeneration rate were significantly higher. Our method for delivering ECMs-enriched stem cells using EFP significantly improved the therapeutic angiogenesis with hADSCs compared to proteolytic enzymatic application.

Enhanced Neovascularization Using Injectable and rhVEGF‐Releasing Cryogel Microparticles

Cryogels are gel networks or scaffolds with a large porous structure; they can be tailored for injectability and for possessing a shape-memory ability. Herein, a growth factor-releasing cryogel microparticle (CMP) system is fabricated, and the therapeutic efficacy of recombinant human vascular endothelial growth factor (rhVEGF)-loaded CMP (V-CMP) for neovascularization is investigated. To prepare the cryogels, both methacrylated chitosan (Chi-MA) and methacrylated chondroitin sulfate (CS-MA) are used, and crosslinking using a radical crosslinking reaction is established. The physical, mechanical, and biological properties of the cryogels are analyzed by varying the amount of CS-MA used. The cryogels are then pulverized, and microsized CMPs are fabricated. CMPs dispersed in saline demonstrate a shear-thinning property, and can thus be extruded through a 23G needle. Additionally, V-CMP exhibit a sustained release profile of rhVEGF and enhance the in vitro proliferation of endothelial cells. Finally, neovascularization and effective tissue necrosis prevention are observed when V-CMPs are injected into a hindlimb ischemia mouse model. Thus, the injectable V-CMP system developed herein demonstrates a high potential utility in various tissue regeneration applications based on cell or growth factor delivery.

Endothelial GABBR2 Regulates Post-ischemic Angiogenesis by Inhibiting the Glycolysis Pathway.

Purpose: Angiogenesis post-ischemia plays an essential role in preventing ischemic damage to tissue by improving the blood recovery. Determining the regulatory mechanism of ischemic angiogenesis, therefore, could provide effective therapeutics for ischemic injury.Materials and Methods: The RNA sequencing (RNA-seq) database was used to predict the association of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) with endothelial-specific expression. The role of GABBR2 in angiogenesis was verified in vitro by downregulating GABBR2 in human umbilical vein endothelial cells (HUVECs) with lentiviral vectors. Besides, the in vivo effect of GABBR2 on the blood recovery of an ischemic hindlimb was demonstrated by establishing a hindlimb ischemia model in normal and GABBR2 adenoviral vector-infected mice. Then, the mobilization of endothelial progenitor cells (EPCs) in peripheral blood post-ischemia was determined by flow cytometry. Finally, the XF analyzer and Western blot were used to determine the effect of GABBR2 on endothelial metabolism.Results: The RNA-seq results indicated a strong association between GABBR2 and endothelial revascularization, and the upregulation of GABBR2 was detected in both hypoxia-treated HUVECs and ischemic mouse hindlimb. Hypoxia treatment for 6 h increased the proliferation, migration, and tube formation of HUVECs, which were inhibited by GABBR2 knockdown. Additionally, GABBR2 downregulation significantly decreased the blood flow recovery of mouse ischemic hindlimb. The expressions of the EPC markers CD34+ and CD133+ significantly decreased in the peripheral blood in hindlimb post-ischemia. Mechanically, glycolysis-dominated metabolism of HUVECs was compromised by GABBR2 knockdown. Evidences of the decreased expressions of HKII, PFKFB3, and PKM1 also supported the compromised glycolysis induced by GABBR2 downregulation.Conclusion: Our study demonstrated that GABBR2 regulated angiogenesis post-ischemia by inhibiting the glycolysis pathway.

MiR-181b suppresses angiogenesis by directly targeting cellular communication network factor 1

Angiogenesis is essential for various physiological and pathological processes. Previous studies have shown that miRNAs play an important role in blood vessel development and angiogenesis. Recent studies have suggested that miR-181b might be involved in the regulation of angiogenesis in tumors. However, whether miR-181b plays a role in angiogenesis in nontumor diseases is unclear. We found that miR-181b expression was downregulated in hypoxia-stimulated primary human umbilical vein endothelial cells (HUVECs) and a mouse hindlimb ischemia (HLI) model. Gain- and loss-of-function studies showed that a miR-181b mimic inhibited HUVEC migration and tube formation in vitro, and a miR-181b inhibitor had the opposite effects. In vivo, agomir-181b suppressed perfusion recovery in the HLI model and capillary density in a Matrigel plug assay, while perfusion recovery and capillary density were increased by injection of antagomir-181b. Mechanistically, we showed with a reporter assay that cellular communication network factor 1 (CCN1) was a direct target of miR-181b. Moreover, miR-181b suppressed angiogenesis at least in part by targeting CCN1 to inhibit the AMPK signaling pathway. Our research suggests that miR-181b suppresses angiogenesis by directly targeting CCN1, which provides new clues for pro-angiogenic treatment strategies.miR-181b inhibits HUVEC migration and tube formation in vitro, and suppresses perfusion recovery in a hindlimb ischemia animal model and in a capillary density assay. Cellular communication network factor 1 (CCN1) is a direct target of miR-181b. miR-181b suppresses angiogenesis at least in part by targeting CCN1 to inhibit the AMPK signaling pathway.