Parenchymal cues define Vegfa-driven venous angiogenesis by activating a sprouting competent venous endothelial subtype.

Formation of organo-typical vascular networks requires cross-talk between differentiating parenchymal cells and developing blood vessels. Here we identify a Vegfa driven venous sprouting process involving parenchymal to vein cross-talk regulating venous endothelial Vegfa signaling strength and subsequent formation of a specialized angiogenic cell, prefabricated with an intact lumen and pericyte coverage, termed L-Tip cell. L-Tip cell selection in the venous domain requires genetic interaction between vascular Aplnra and Kdrl in a subset of venous endothelial cells and exposure to parenchymal derived Vegfa and Apelin. Parenchymal Esm1 controls the spatial positioning of venous sprouting by fine-tuning local Vegfa availability. These findings may provide a conceptual framework for understanding how Vegfa generates organo-typical vascular networks based on the selection of competent endothelial cells, induced via spatio-temporal control of endothelial Kdrl signaling strength involving multiple parenchymal derived cues generated in a tissue dependent metabolic context.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

MiR-522-3p Attenuates Cardiac Recovery by Targeting FOXP1 to Suppress Angiogenesis.

Angiogenesis is crucial for blood supply reconstitution after myocardial infarction in patients with acute coronary syndrome (ACS). MicroRNAs are recognized as important epigenetic regulators of endothelial angiogenesis. The purpose of this study is to determine the roles of miR-522-3p in angiogenesis after myocardial infarction. The expression levels of miR-522-3p in rats' plasma and in the upper part of the ligation of the heart tissues at 28 days after myocardial infarction were significantly higher than those of the sham group. miR-522-3p mimics inhibited cell proliferations, migrations, and tube formations under hypoxic conditions in HUVECs (human umbilical vein endothelial cells), whereas miR-522-3p inhibitors did the opposite. Furthermore, studies have indicated that the inhibition of miR-522-3p by antagomir infusion promoted angiogenesis and accelerated the recovery of cardiac functions in rats with myocardial infarction.Data analysis and experimental results revealed that FOXP1 (Forkhead-box protein P1) was the target gene of miR-522-3p. Our study explored the mechanism of cardiac angiogenesis after myocardial infarction and provided a potential therapeutic approach for the treatment of ischemic heart disease in the future.

Soft Tissue Reconstruction.

Autologous fat transplantation has revolutionized soft tissue reconstruction, but conventional methods remain unpredictable as graft resorption rates are high due to lack of vascularization. The advent of adipose-derived stem cells (ASCs) has led to improvement of fat grafting outcomes, in part to their ability to undergo facile differentiation into adipose tissue, their angiogenic properties, and their ability to express and secrete multiple growth factors. This chapter discusses the isolation and characterization of human ASCs, its expansion in vitro, and relevant in vivo models for adipose tissue engineering.

Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment.

The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

LncRNA NEAT1/miR-146a-5p Axis Restores Normal Angiogenesis in Diabetic Foot Ulcers by Targeting mafG.

Non-healing lesions in diabetic foot ulcers are a significant effect of poor angiogenesis. Epigenetic regulators, mainly lncRNA and miRNA, are recognized for their important roles in disease progression. We deciphered the regulation of lncRNA NEAT1 through the miR-146a-5p/mafG axis in the progression of DFU. A lowered expression of lncRNA NEAT1 was associated with dysregulated angiogenesis through the reduced expression of mafG, SDF-1α, and VEGF in chronic ulcer subjects compared to acute DFU. This was validated by silencing NEAT1 by SiRNA in the endothelial cells which resulted in the transcriptional repression of target genes. Our in silico analysis identified miR-146a-5p as a potential target of lncRNA NEAT1. Further, silencing NEAT1 led to an increase in the levels of miR-146a-5p in chronic DFU subjects. This research presents the role of the lncRNA NEAT1/miR-146a-5p/mafG axis in enhancing angiogenesis in DFU.

Recent advances in endothelial colony-forming cells: from the transcriptomic perspective.

Endothelial colony-forming cells (ECFCs) are progenitors of endothelial cells with significant proliferative and angiogenic ability. ECFCs are a promising treatment option for various diseases, such as ischemic heart disease and peripheral artery disease. However, some barriers hinder the clinical application of ECFC therapeutics. One of the current obstacles is that ECFCs are dysfunctional due to the underlying disease states. ECFCs exhibit dysfunctional phenotypes in pathologic states, which include but are not limited to the following: premature neonates and pregnancy-related diseases, diabetes mellitus, cancers, haematological system diseases, hypoxia, pulmonary arterial hypertension, coronary artery diseases, and other vascular diseases. Besides, ECFCs are heterogeneous among donors, tissue sources, and within cell subpopulations. Therefore, it is important to elucidate the underlying mechanisms of ECFC dysfunction and characterize their heterogeneity to enable clinical application. In this review, we summarize the current and potential application of transcriptomic analysis in the field of ECFC biology. Transcriptomic analysis is a powerful tool for exploring the key molecules and pathways involved in health and disease and can be used to characterize ECFC heterogeneity.

Exosomal hsa_circ_0093884 derived from endothelial progenitor cells promotes therapeutic neovascularization via miR-145/SIRT1 pathway.

Therapeutic neovascularization is a strategy to promote blood vessel growth and improve blood flow, which is critical to tissue repair and regeneration in ischemic diseases. Here, we investigated the role of endothelial progenitor cell - derived exosomes (EPC-Exos) in therapeutic neovascularization and clarified the mechanism of hsa_circ_0093884 in EPC-Exos mediated neovascularization. Injection of EPC-Exos improved mouse ischemic hindlimb perfusion, promoted angiogenesis in Matrigel plugs and mouse skin wound healing. In vitro coculture with EPC-Exos improved HUVEC proliferation, angiogenic and migration ability, while alleviated hypoxia-induced apoptosis. hsa_circ_0093884 was identified from eleven types of circRNA derived from SIRT1 and proved to be enriched in EPC-Exos. Overexpression of hsa_circ_0093884 in EPC-Exos further enhanced the angiogenic capacity, while knockdown of hsa_circ_0093884 abolished the benefits. Mechanistically, EPC-Exos mediated shuttling of hsa_circ_0093884 induced cytoplasmic sponge of miR-145, thereby releasing repression of SIRT1. In vitro co-transfection indicated silence of miR-145 further strengthened the angiogenic effect of hsa_circ_0093884, while overexpression of miR-145 inhibited hsa_circ_0093884 mediated angiogenesis and abolished the beneficial effect of EPC-Exos. Furthermore, in vivo experiments using endothelial specific SIRT1 conditional knockout mice indicated hsa_circ_0093884 overexpressing EPC-Exos failed to promote therapeutic neovascularization in SIRT1cKO mice. Collectively, our results demonstrated that EPC-Exos promoted therapeutic neovascularization through hsa_circ_0093884/miR-145/SIRT1 axis.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

Branched Channels in Porous ß-Tricalcium Phosphate Scaffold Promote Vascularization.

Rapid and efficient vascularization is still considerably challenging for a porous ß-tricalcium phosphate (ß-TCP) scaffold to achieve. To overcome this challenge, branched channels were created in the porous ß-TCP scaffold by using 3D printing and a template-casting method to facilitate the instant flow of blood supply. Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were seeded in the channeled porous scaffolds and characterized through a double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay, and cell migration. Channeled porous ß-TCP scaffolds were then implanted in the subcutaneous pockets of mice. Histological staining and immunohistochemical staining on vascularization and bone-related markers were carried out on the embedded paraffin sections. Results from in vitro experiments showed that branched channels significantly promoted HUVECs' infiltration, migration, proliferation, and angiogenesis, and also promoted the proliferation and osteogenesis differentiation of hBMSCs. In vivo implantation results showed that, in the early stage after implantation, cells significantly migrated into branched channeled scaffolds. More matured blood vessels formed in the branched channeled scaffolds compared to that in nonchanneled and straight channeled scaffolds. Beside promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous ß-TCP scaffold promoted rapid vascularization and potentially stimulated bone cells recruitment.

Calcitonin gene-related peptide-modulated macrophage phenotypic alteration regulates angiogenesis in early bone healing.

OBJECTIVES: This study aimed to investigate the effect of calcitonin gene-related peptide (CGRP) on the temporal alteration of macrophage phenotypes and macrophage-regulated angiogenesis duringearlybonehealing and preliminarily elucidate the mechanism. METHODS: In vivo, the rat mandibular defect models were established with inferior alveolar nerve transection (IANT) or CGRP receptor antagonist injection. Radiographicandhistologic assessments for osteogenesis, angiogenesis, and macrophage phenotypic alteration within bone defects were performed. In vitro, the effect and mechanism of CGRP on macrophage polarization and phenotypic alteration were analyzed. Then the conditioned medium (CM) from CGRP-treated M1 or M2 macrophages was used to culture human umbilical vein endothelial cells (HUVECs), and the CGRP's effect on macrophage-regulated angiogenesis was detected. RESULTS: Comparable changes following IANT and CGRP blockade within bone defects were observed, including the suppression of early osteogenesis and angiogenesis, the prolonged M1 macrophage infiltration and the prohibited transition toward M2 macrophages around vascular endothelium. In vitro experiments showed that CGRP promoted M2 macrophage polarization while upregulating the expression of interleukin 6 (IL-6), a major cytokine that facilitates the transition from M1 to M2-dominant stage, in M1 macrophages via the activation of Yes-associated protein 1. Moreover, CGRP-treated macrophage-CM showed an anabolic effect on HUVECs angiogenesis compared with macrophage-CM and might prevail over the direct effect of CGRP on HUVECs. CONCLUSIONS: Collectively, our results reveal the effect of CGRP on M1 to M2 macrophage phenotypic alteration possibly via upregulating IL-6 in M1 macrophages, and demonstrate the macrophage-regulated pro-angiogenic potential of CGRP in early bone healing.

Engineering primitive multiscale chimeric vasculature by combining human microvessels with explanted murine vessels.

Strategies to separately manufacture arterial-scale tissue engineered vascular grafts and microvascular networks have been well-established, but efforts to bridge these two length scales to create hierarchical vasculature capable of supporting parenchymal cell functions or restoring perfusion to ischemic tissues have been limited. This work aimed to create multiscale vascular constructs by assessing the capability of macroscopic vessels isolated from mice to form functional connections to engineered capillary networks ex vivo. Vessels of venous and arterial origins from both thoracic and femoral locations were isolated from mice, and then evaluated for their abilities to sprout endothelial cells (EC) capable of inosculating with surrounding human cell-derived microvasculature within bulk fibrin hydrogels. Comparing aortae, vena cavae, and femoral vessel bundles, we identified the thoracic aorta as the rodent macrovessel that yielded the greatest degree of sprouting and interconnection to surrounding capillaries. The presence of cells undergoing vascular morphogenesis in the surrounding hydrogel attenuated EC sprouting from the macrovessel compared to sprouting into acellular hydrogels, but ultimately sprouted mouse EC interacted with human cell-derived capillary networks in the bulk, yielding chimeric vessels. We then integrated micromolded mesovessels into the constructs to engineer a primitive 3-scale vascular hierarchy comprising capillaries, mesovessels, and macrovessels. Overall, this study yielded a primitive hierarchical vasculature suitable as proof-of-concept for regenerative medicine applications and as an experimental model to better understand the spontaneous formation of host-graft vessel anastomoses.

Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

Human lens epithelial-secreted exosomes attenuate ocular angiogenesis via inhibiting microglial activation.

The lens is an avascular tissue, where epithelial cells (LECs) are the primary living cells. The role of LECs-derived exosomes (LEC-exos) is largely unknown. In our study, we determined the anti-angiogenic role of LEC-exos, manifested as regressed retinal neovascularization (NV) using the oxygen-induced retinopathy (OIR), and reduced choroidal NV size and pathological vascular leakage using the laser-induced choroidal neovascularization (laser-induced CNV). Furthermore, the activation and accumulation of microglia were also restricted by LEC-exos. Based on Luminex multiplex assays, the expressions of chemokines such as SCYB16/CXCL16, MCP-1/CCL2, I-TAC/CXCL11, and MIP 3beta/CCL19 were decreased after treatment with LEC-exos. Transwell assays showed that LEC-exos restricted the migration of the mouse microglia cell line (BV2 cells). After incubation with LEC-exos-treated BV2 cells, human umbilical vein endothelial cells (hUVECs) were collected for further evaluation using tube formation, Transwell assays, and 5-ethynyl-2'-deoxyuridine (EDU) assays. Using in vitro experiments, the pro-angiogenic effect of microglia was restricted by LEC-exos. Hence, it was investigated that LEC-exos attenuated ocular NV, which might attribute to the inhibition of microglial activation and accumulation.

Electrical stimulation promoting the angiogenesis in diabetic rat perforator flap through attenuating oxidative stress-mediated inflammation and apoptosis.

Background: Skin flap transplantation is one of the effective methods to treat the diabetes-related foot ulceration, but the intrinsic damage to vessels in diabetes mellitus (DM) leads to the necrosis of skin flaps. Therefore, the discovery of a non-invasive and effective approach for promoting the survival of flaps is of the utmost importance. Electrical stimulation (ES) promotes angiogenesis and increases the proliferation, migration, and elongation of endothelial cells, thus being a potential effective method to improve flap survival. Objective: The purpose of this study was to elucidate the mechanism used by ES to effectively restore the impaired function of endothelial cells caused by diabetes. Methods: A total of 79 adult male Sprague-Dawley rats were used in this study. Gene and protein expression was assessed by PCR and western blotting, respectively. Immunohistochemistry and hematoxylin-eosin staining were performed to evaluate the morphology and density of the microvessels in the flap. Results: The optimal duration for preconditioning the flap with ES was 7 days. The flap survival area percentage and microvessels density in the DMES group were markedly increased compared to the DM group. VEGF, MMP2, and MMP9 protein expression was significantly upregulated. ROS intensity was significantly decreased and GSH concentration was increased. The expression of IL-1ß, MCP­1, cleaved caspase-3, and Bax were downregulated in the DMES group, while TGF-ß expression was upregulated. Conclusions: ES improves the angiogenesis in diabetic ischemic skin flaps by attenuating oxidative stress-mediated inflammation and apoptosis, eventually increasing their viability.

Review: Human stem cell-based 3D in vitro angiogenesis models for preclinical drug screening applications.

Vascular diseases are the underlying pathology in many life-threatening illnesses. Human cellular and molecular mechanisms involved in angiogenesis are complex and difficult to study in current 2D in vitro and in vivo animal models. Engineered 3D in vitro models that incorporate human pluripotent stem cell (hPSC) derived endothelial cells (ECs) and supportive biomaterials within a dynamic microfluidic platform provide a less expensive, more controlled, and reproducible platform to better study angiogenic processes in response to external chemical or physical stimulus. Current studies to develop 3D in vitro angiogenesis models aim to establish single-source systems by incorporating hPSC-ECs into biomimetic extracellular matrices (ECM) and microfluidic devices to create a patient-specific, physiologically relevant platform that facilitates preclinical study of endothelial cell-ECM interactions, vascular disease pathology, and drug treatment pharmacokinetics. This review provides a detailed description of the current methods used for the directed differentiation of human stem cells to endothelial cells and their use in engineered 3D in vitro angiogenesis models that have been developed within the last 10 years.

The comparison of adipose-derived stromal cells (ADSCs) delivery method in a murine model of hindlimb ischemia.

BACKGROUND: Adipose-derived stromal cells (ADSCs) demonstrate ability to promote tissue healing and down-regulate excessive inflammation. ADSCs have been used to treat critical limb ischemia in preclinical and clinical trials, but still, there is little known about their optimal delivery strategy. To date, no direct analysis of different methods of ADSCs delivery has been performed in the hindlimb ischemia model. Therefore, in this study we focused on the therapeutic efficacy of different ADSCs delivery methods in a murine model of hindlimb ischemia. METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hindlimb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6. ADSCs were delivered directly into ischemic muscle, into the contralateral muscle or intravenously. 7 and 14 days after the surgery, the gastrocnemius and quadriceps muscles were collected for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: Our research revealed that muscle regeneration, angiogenesis, arteriogenesis and macrophage infiltration in murine model of hindlimb ischemia differ depending on ADSCs delivery method. We have demonstrated that intramuscular method (directly into ischemic limb) of ADSCs delivery is more efficient in functional recovery after critical limb ischemia than intravenous or contralateral route. CONCLUSIONS: We have noticed that injection of ADSCs directly into ischemic limb is the optimal delivery strategy because it increases: (1) muscle fiber regeneration, (2) the number of capillaries and (3) the influx of macrophages F4/80+/CD206+.

Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced ß-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.