FGF2-primed 3D spheroids producing IL-8 promote therapeutic angiogenesis in murine hindlimb ischemia.
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ABSTRACT:
SUBMITTER: Choi J
PROVIDER: S-EPMC8373896 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Critical limb ischemia (CLI) is associated with a higher risk of limb amputation and cardiovascular death. Dapagliflozin has shown great potential in the treatment of cardiovascular disease. However, the effects of dapagliflozin on CLI and the underlying mechanisms have not been fully elucidated. We evaluated the effect of dapagliflozin on recovery from limb ischemia using a mouse model of hindlimb ischemia. The flow of perfusion was evaluated using a laser Doppler system. Tissue response was assessed by analyzing capillary density, arterial density, and the degree of fibrosis in the gastrocnemius muscle. Immunofluorescence and Western blot were used to detect the expression of macrophage polarization markers and inflammatory factors. Our findings demonstrate the significant impact of dapagliflozin on the acceleration of blood flow recovery in a hindlimb ischemia mouse model, concomitant with a notable reduction in limb necrosis. Histological analysis revealed that dapagliflozin administration augmented the expression of key angiogenic markers, specifically CD31 and α-SMA, while concurrently mitigating muscle fibrosis. Furthermore, our investigation unveiled dapagliflozin's ability to induce a phenotypic shift of macrophages from M1 to M2, thereby diminishing the expression of inflammatory factors, including IL-1β, IL-6, and TNF-α. These effects were partially mediated through modulation of the NF-κB signaling pathway. Lastly, we observed that endothelial cell proliferation, migration, and tube-forming function are enhanced in vitro by utilizing a macrophage-conditioned medium derived from dapagliflozin treatment. Taken together, our study provides evidence that dapagliflozin holds potential as an efficacious therapeutic intervention in managing CLI by stimulating angiogenesis, thereby offering a novel option for clinical CLI treatment.
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Project description:Interleukin (IL) 35 is a novel immunosuppressive heterodimeric cytokine in IL-12 family. Whether and how IL-35 regulates ischemia-induced angiogenesis in peripheral artery diseases are unrevealed. To fill this important knowledge gap, we used loss-of-function, gain-of-function, omics data analysis, RNA-Seq, in vivo and in vitro experiments, and we have made the following significant findings: i) IL-35 and its receptor subunit IL-12RB2, but not IL-6ST, are induced in the muscle after hindlimb ischemia (HLI); ii) HLI-induced angiogenesis is improved in Il12rb2-/- mice, in ApoE-/-/Il12rb2-/- mice compared to WT and ApoE-/- controls, respectively, where hyperlipidemia inhibits angiogenesis in vivo and in vitro; iii) IL-35 cytokine injection as a gain-of-function approach delays blood perfusion recovery at day 14 after HLI; iv) IL-35 spares regenerative angiogenesis at the late phase of HLI recovery after day 14 of HLI; v) Transcriptome analysis of endothelial cells (ECs) at 14 days post-HLI reveals a disturbed extracellular matrix re-organization in IL-35-injected mice; vi) IL-35 downregulates three reactive oxygen species (ROS) promoters and upregulates one ROS attenuator, which may functionally mediate IL-35 upregulation of anti-angiogenic extracellular matrix proteins in ECs; and vii) IL-35 inhibits human microvascular EC migration and tube formation in vitro mainly through upregulating anti-angiogenic extracellular matrix-remodeling proteins. These findings provide a novel insight on the future therapeutic potential of IL-35 in suppressing ischemia/inflammation-triggered inflammatory angiogenesis at early phase but sparing regenerative angiogenesis at late phase.
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Project description:The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis.
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Project description:Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8-12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31+/BrdU+ cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO+ cells), NETs (MPO+/CitH3+/DAPI+), and macrophages (CD68+ cells) in SV-129 mice, whereas macrophage polarization (MRC1- vs. MRC1+) and total leukocyte infiltration (CD45+ cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.
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Project description:Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10(7)) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc(+)-eGFP(+), mADSCs(Fluc+GFP+)), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells' distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.
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Project description:Therapeutic strategies using endogenous stem cell mobilizer can provide effective cell-free therapy for addressing various ischemic diseases. In particular, substance P (SP) exhibited therapeutic regeneration by facilitating mobilization of endogenous stem cells from bone marrow to the injured sites. However, its therapeutic effect has been limited due to short half-life and rapid degradation of administered SP peptides in vivo. Here we sought to develop high-density lipoprotein (HDL)-mimicking nanodiscs conjugated with SP (HDL-SP) in order to increase the in vivo half-life, bone marrow targeting, and therapeutic efficacy of SP for the treatment of diabetic peripheral ischemia. Conjugation of SP onto HDL nanodisc led to remarkable ∼3215- and ∼1060-fold increase in the ex vivo and in vivo half-lives of SP, respectively. Accordingly, HDL-SP nanodiscs improved retention of SP in bone marrow after systemic administration, leading to efficient mobilization of stem cells from bone marrow into blood circulation and reduction of systemic inflammation. Consequently, nanodisc based SP peptide delivery promoted blood vessel formation, blood perfusion recovery and markedly improved limb salvage in diabetic hindlimb ischemia model relative to administration of free SP without nanodisc modification. Therefore, HDL-SP nanodisc can provide a novel strategy for the treatment of diabetic ischemia and HDL nanodisc modification could be potentially useful for the extension of plasma circulation of other labile peptides.
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Project description:With critical limb ischemia (CLI) being a multi-factorial disease, it is becoming evident that gene therapy with a multiple bio-functional growth factor could achieve better therapeutic outcomes. Cytochrome P450 epoxygenase-2J2 (CYP2J2) and its catalytic products epoxyeicosatrienoic acids (EETs) exhibit pleiotropic biological activities, including pro-angiogenic, anti-inflammatory and cardiovascular protective effects, which are considerably beneficial for reversing ischemia and restoring local blood flow in CLI. Here, we designed a nanoparticle-based pcDNA3.1-CYP2J2 plasmid DNA (pDNA) delivery system (nanoparticle/pDNA complex) composed of a novel three-arm star block copolymer (3S-PLGA-po-PEG), which was achieved by conjugating three-armed PLGA to PEG via the peroxalate ester bond. Considering the multiple bio-functions of CYP2J2-EETs and the sensitivity of the peroxalate ester bond to H2O2, this nanoparticle-based gene delivery system is expected to exhibit excellent pro-angiogenic effects while improving the high oxidative stress and inflammatory micro-environment in ischemic hindlimb. Our study reports the first application of CYP2J2 in the field of therapeutic angiogenesis for CLI treatment and our findings demonstrated good biocompatibility, stability and sustained release properties of the CYP2J2 nano-delivery system. In addition, this nanoparticle-based gene delivery system showed high transfection efficiency and efficient VEGF expression in vitro and in vivo. Intramuscular injection of nanoparticle/pDNA complexes into mice with hindlimb ischemia resulted in significant rapid blood flow recovery and improved muscle repair compared to mice treated with naked pDNA. In summary, 3S-PLGA-po-PEG/CYP2J2-pDNA complexes have tremendous potential and provide a practical strategy for the treatment of limb ischemia. Moreover, 3S-PLGA-po-PEG nanoparticles might be useful as a potential non-viral carrier for other gene delivery applications.
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Project description:The promise of cell therapy for repair and restoration of damaged tissues or organs relies on administration of large dose of cells whose healing benefits are still limited and sometimes irreproducible due to uncontrollable cell loss and death at lesion sites. Using a large amount of therapeutic cells increases the costs for cell processing and the risks of side effects. Optimal cell delivery strategies are therefore in urgent need to enhance the specificity, efficacy, and reproducibility of cell therapy leading to minimized cell dosage and side effects. Here, we addressed this unmet need by developing injectable 3D microscale cellular niches (microniches) based on biodegradable gelatin microcryogels (GMs). The microniches are constituted by in vitro priming human adipose-derived mesenchymal stem cells (hMSCs) seeded within GMs resulting in tissue-like ensembles with enriched extracellular matrices and enhanced cell-cell interactions. The primed 3D microniches facilitated cell protection from mechanical insults during injection and in vivo cell retention, survival, and ultimate therapeutic functions in treatment of critical limb ischemia (CLI) in mouse models compared with free cell-based therapy. In particular, 3D microniche-based therapy with 10(5) hMSCs realized better ischemic limb salvage than treatment with 10(6) free-injected hMSCs, the minimum dosage with therapeutic effects for treating CLI in literature. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy for treating CLI with the lowest cell dosage in mouse models. This study offers a widely applicable cell delivery platform technology to boost the healing power of cell regenerative therapy.
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Project description:Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)-based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti-human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti-CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.
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