Mouse Hindlimb Ischemia Research Articles

Subaqueous 3D stem cell spheroid levitation culture using anti-gravity bioreactor based on sound wave superposition

BackgroundRecently, various studies have revealed that 3D cell spheroids have several advantages over 2D cells in stem cell culture. However, conventional 3D spheroid culture methods have some disadvantages and limitations such as time required for spheroid formation and complexity of the experimental process. Here, we used acoustic levitation as cell culture platform to overcome the limitation of conventional 3D culture methods.MethodsIn our anti-gravity bioreactor, continuous standing sonic waves created pressure field for 3D culture of human mesenchymal stem cells (hMSCs). hMSCs were trapped and aggerated in pressure field and consequently formed spheroids. The structure, viability, gene and protein expression of spheroids formed in the anti-gravity bioreactor were analyzed by electron microscope, immunostaining, polymerase chain reaction, and western blot. We injected hMSC spheroids fabricated by anti-gravity bioreactor into the mouse hindlimb ischemia model. Limb salvage was quantified to evaluate therapeutic efficacy of hMSC spheroids.ResultsThe acoustic levitation in anti-gravity bioreactor made spheroids faster and more compact compared to the conventional hanging drop method, which resulted in the upregulation of angiogenic paracrine factors of hMSCs, such as vascular endothelial growth factor and angiopoietin 2. Injected hMSCs spheroids cultured in the anti-gravity bioreactor exhibited improved therapeutic efficacy, including the degree of limb salvage, capillary formation, and attenuation of fibrosis and inflammation, for mouse hindlimb ischemia model compared to spheroids formed by the conventional hanging drop method.ConclusionOur stem cell culture system using acoustic levitation will be proposed as a new platform for the future 3D cell culture system.Graphical

Extracellular vesicles derived from hypoxia-preconditioned bone marrow mesenchymal stem cells ameliorate lower limb ischemia by delivering miR-34c.

Hypoxic mesenchymal stem cell-derived extracellular vesicles (EVs) have been suggested as a promising therapy for various diseases. This study aims to determine the effect of EVs derived from bone marrow mesenchymal stem cells (BMMSCs) under hypoxia on lower limb ischemia and the underlying mechanism. Human BMMSCs were subjected to hypoxia or normoxia followed by the isolation of EVs. Nanoparticle trafficking analysis (NTA), transmission electron microscopy (TEM), and Western Blotting using corresponding markers were performed to confirm the EVs. The EVs from BMMSCs under hypoxia condition (Hyp-EVs) or normoxia condition (Nor-EVs) were subjected to hindlimb ischemia (HI) mice. MiR-34c expression in BMMSCs and BMMSC-EVs was detected. The role of miR-34c in regulating M2 macrophage polarization, as well as the target of miR-34c, were explored. HI mice with Hyp-EV treatment, as compared to the Nor-EV or the PBS group, had better blood flow and higher capillary density. MiR-34c expression was increased in BMMSCs, BMMSC-EVs, and the adductor muscle of HI mice. Hyp-EVs promoted the M2 macrophage polarization and anti-inflammatory cytokine production, and enhanced the blood flow and capillary density in HI mice, while the knockdown of miR-34c partly reversed these effects. PTEN is a target of miR-34c, and the PTEN silencing facilitated M2 macrophage polarization, whereas the inhibition of AKT signaling partly abolished the effect. Hyp-EVs promoted M2 macrophage polarization by delivering miR-34c via PTEN/AKT pathway, which could be a promising therapeutic strategy to ameliorate lower limb ischemia.

Fibrinogen-based cell and spheroid sheets manipulating and delivery for mouse hindlimb ischemia

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higher in vitro tubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research.

Salidroside facilitates therapeutic angiogenesis in diabetic hindlimb ischemia by inhibiting ferroptosis

Hindlimb ischemia (HLI), in which blood perfusion to the hindlimb is obstructed, is one of the major complications of diabetes. Skeletal muscle cells are crucial for revascularization as they can secrete various angiogenic factors; however, hyperglycemia impairs their viability and subsequently their angiogenic potential. Salidroside can promote skeletal muscle cell viability under hyperglycemia; however, the molecular mechanism is still poorly understood. Here we revealed that salidroside could suppress hyperglycemia-induced ferroptosis in skeletal muscle cells by promoting GPX4 expression, thereby restoring their viability and paracrine functions. These in turn promoted the proliferation and migration potentials of blood vessel-forming cells. Furthermore, we showed that salidroside/GPX4-mediated ferroptosis inhibition is crucial for promoting angiogenesis and blood perfusion recovery in diabetic HLI mice. Together, we reveal a novel molecular mechanism of salidroside in enhancing skeletal muscle cells-mediated revascularization and blood perfusion recovery in diabetic HLI mice, further highlighting it as a potential compound for treating diabetic HLI.

PER1 promotes functional recovery of mice with hindlimb ischemia by inducing anti-inflammatory macrophage polarization

Hindlimb ischemia (HLI) is an arterial occlusive disease that exposes the patients to the risk of limb gangrene and loss. Polarization of macrophages is related to HLI-induced inflammation. Period circadian regulator 1 (PER1) is a core component of the circadian clock. We first showed, based upon bioinformatics analysis of microarray data, that PER1 expression was reduced in monocytes from patients with critical limb ischemia. The proximal femoral artery in the left hindlimb of male mice was ligated and then the femoral artery and its collateral branches were removed to establish the HLI mouse model. After modeling, a single intramuscular injection of 1 × 109 pfu Ad-PER1 was performed at the adductor and gastrocnemius muscles. The gastrocnemius muscle tissues were collected at day 0, 3, 7, 14, 21 post-HLI. There was obvious pathological necrosis, accompanied with reduced expression of PER1 in the muscle tissues of HLI mice. Expression of CD68 and CD31 seemed to be corresponded to PER1 in gastrocnemius muscle, implying the potential of PER1 in regulating macrophage-related inflammation and angiogenesis. PER1 overexpression diminished myocyte damage, promoted blood flow restoration and improved behavioral scores of HLI mice. Immunostaining of CD31 and α-SMA revealed that PER1 upregulation reversed HLI-induced decreases in capillary and arteriole density. In vitro, RAW264.7 cells were cultured in hypoxia (1% O2) for 24 h. The percentage of pro-inflammatory CD86+ macrophages (M1 type) was decreased and that of anti-inflammatory CD206+ macrophages (M2 type) was increased when PER1 was overexpressed. Moreover, the expression levels of TNF-α, IL-6 and M1-type marker iNOS were decreased, and levels of IL-10 and M2-type marker Arg-1 were increased by PER1 in gastrocnemius muscle of HLI mice and hypoxia-treated RAW264.7 cells. PER1 might reduce M1 macrophage polarization and promote M2 macrophage polarization, and thus exert anti-inflammatory and pro-angiogenic actions. Our findings suggest that PER1 overexpression promotes functional recovery of mice with HLI through regulating macrophage polarization.

Effects of Pemafibrate on Reducing Oxidative Stress and Augmenting Angiogenesis in Ischemic Limb Tissue.

Oxidative damage is observed in the ischemic limbs of patients with arteriosclerosis obliterans. We investigated whether pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, reduced oxidative stress in ischemic limbs and consequently rescued limb damage in model mice. We surgically induced hind-limb ischemia in mice and orally administered pemafibrate solution (P-05 group, 0.5 mg/kg/day; P-10 group, 1.0 mg/kg/day) or control solution (control group). Seven days after the surgery, differences in reactive oxygen species (ROS) contents, antioxidative enzyme and transcription factor expression, blood flow, and capillary density in ischemic limbs were assessed. Tissue ROS levels were lower in the P-05 and P-10 groups compared with those in the control group. Although the tissue expression levels of nuclear factor-erythroid 2-related factor 2 increased in the P-10 group compared with that in the control group, no corresponding changes were observed in the tissue expression of four antioxidative enzymes. The limb salvage rates and capillary densities in ischemic limbs were higher in the P-05 and P-10 groups than that in the control group. Pemafibrate treatment reduced oxidative stress and augmented angiogenesis in ischemic limbs, contributing to prevention of limb damage in mice.

SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis.

Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1-40 μM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 μM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 μM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.

GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression

Regulatory T cells (Tregs) are critically involved in neovascularization, an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174), which is a regulator of Treg function and development, in neovascularization remains elusive. Here, we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs, thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically, GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via Gαs/cAMP/PKA signal pathway activation. Collectively, these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases.

ENO1 expression and Erk phosphorylation in PDAC and their effects on tumor cell apoptosis in a hypoxic microenvironment.

Hypoxia is an important feature of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that hypoxia promotes ENO1 expression and PDAC invasion. However, the underlying molecular mechanism was remains unclear. The relationship between ENO1 expression and clinicopathological characteristics was analyzed in 84 patients with PADC. The effects of CoCl2-induced hypoxia and ENO1 downregulation on the apoptosis, invasion, and proliferation of PDAC cells were evaluated in vitro and in vivo. Hypoxia- and ENO1-induced gene expression was analyzed by transcriptomic sequencing. The prognosis of PDAC with high ENO1 expression was poor (P < 0.05). High ENO1 expression was closely associated with histological differentiation and tumor invasion in 84 PDAC cases (P < 0.05). Hypoxia increased ENO1 expression in PDAC and promoted its migration and invasion. Apoptotic cells and the apoptosis marker caspase-3 in the CoCl2-treated ENO1-sh group were significantly elevated (P < 0.05). Transcriptomic sequencing indicated that CoCl2-induced PDAC cells initiated MAPK signaling. Under hypoxic conditions, PDAC cells upregulated ENO1 expression, thereby accelerating ERK phosphorylation and inhibiting apoptosis (P < 0.05). Consistent results were also observed in a PDAC-bearing mouse hindlimb ischemia model. Hypoxia-induced ENO1 expression promotes ERK phosphorylation and inhibits apoptosis, thus leading to PDAC survival and invasion. These results suggest that ENO1 is a potential therapeutic target for PDAC.

Repair of Limb Ischemia Is Dependent on Hematopoietic Stem Cell Specific-SHP-1 Regulation of TGF-β1.

Hematopoietic stem cell (HSC) therapy has shown promise for tissue regeneration after ischemia. Therefore, there is a need to understand mechanisms underlying endogenous HSCs activation in response to ischemic stress and coordination of angiogenesis and repair. SHP-1 plays important roles in HSC quiescence and differentiation by regulation of TGF-β1 signaling. TGF-β1 promotes angiogenesis by stimulating stem cells to secrete growth factors to initiate the formation of blood vessels and later aid in their maturation. We propose that SHP-1 responds to ischemia stress in HSC and progenitor cells (HSPC) via regulation of TGF-β1. A mouse hind limb ischemia model was used. Local blood perfusion in the limbs was determined using laser doppler perfusion imaging. The number of positive blood vessels per square millimeter, as well as blood vessel diameter (μm) and area (μm2), were calculated. Hematopoietic cells were analyzed using flow cytometry. The bone marrow transplantation assay was performed to measure HSC reconstitution. After femoral artery ligation, TGF-β1 was initially decreased in the bone marrow by day 3 of ischemia, followed by an increase on day 7. This pattern was opposite to that in the peripheral blood, which is concordant with the response of HSC to ischemic stress. In contrast, SHP-1 deficiency in HSC is associated with irreversible activation of HSPCs in the bone marrow and increased circulating HSPCs in peripheral blood following limb ischemia. In addition, there was augmented auto-induction of TGF-β1 and sustained inactivation of SHP-1-Smad2 signaling, which impacted TGF-β1 expression in HSPCs in circulation. Importantly, restoration of normal T GF-β1 oscillations helped in the recovery of limb repair and function. HSPC-SHP-1-mediated regulation of TGF-β1 in both bone marrow and peripheral blood is required for a normal response to ischemic stress.

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

MicroRNA-29a Regulates ADAM12 Through Direct Interaction With ADAM12 mRNA and Modulates Postischemic Perfusion Recovery.

BackgroundPeripheral artery disease is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the postischemic adaptation involved in restoring blood flow in peripheral artery disease. Previously, in a murine hind limb ischemia model of peripheral artery disease, we identified ADAM12 (a disintegrin and metalloproteinase gene 12) as a key genetic modifier of postischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MicroRNAs are a class of small, noncoding, single‐stranded RNAs that regulate gene expression primarily through transcriptional repression of messenger RNA (mRNA). We showed microRNA‐29a (miR‐29a) modulates ADAM12 expression in the setting of diabetes and ischemia. However, how miR‐29a modulates ADAM12 is not known. Moreover, the physiological effects of miR‐29a modulation in a nondiabetic setting is not known.Methods and ResultsWe overexpressed or inhibited miR‐29a in ischemic mouse gastrocnemius and tibialis anterior muscles, and quantified the effect on perfusion recovery, ADAM12 expression, angiogenesis, and skeletal muscle regeneration. In addition, using RNA immunoprecipitation–based anti‐miR competitive assay, we investigated the interaction of miR‐29a and ADAM12 mRNA in mouse microvascular endothelial cell, skeletal muscle, and human endothelial cell lysates. Ectopic expression of miR‐29a in ischemic mouse hind limbs decreased ADAM12 mRNA expression, increased skeletal muscle injury, decreased skeletal muscle function, and decreased angiogenesis and perfusion recovery, with no effect on skeletal muscle regeneration and myofiber cross‐sectional area following hind limb ischemia. RNA immunoprecipitation–based anti‐miR competitive assay studies showed miR‐29a antagomir displaced miR‐29a and ADAM12 mRNA from the AGO‐2 (Argonaut‐2) complex in a dose dependent manner.ConclusionsTaken together, the data show miR‐29a suppresses ADAM12 expression by directly binding to its mRNA, resulting in impaired skeletal muscle function, angiogenesis, and poor perfusion. Hence, elevated levels of miR‐29a, as seen in diabetes and aging, likely contribute to vascular pathology, and modulation of miR‐29a could be a therapeutic target.

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia.

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.

Endothelium-targeted delivery of PPARδ by adeno-associated virus serotype 1 ameliorates vascular injury induced by hindlimb ischemia in obese mice

Diabetic vasculopathy is a major health problem worldwide. Peripheral arterial disease (PAD), and in its severe form, critical limb ischemia is a major form of diabetic vasculopathy with limited treatment options. Existing literature suggested an important role of PPARδ in vascular homeostasis. It remains elusive for using PPARδ as a potential therapeutic target due to mostly the side effects of PPARδ agonists. To explore the roles of PPARδ in endothelial homeostasis, endothelial cell (EC) selective Ppard knockout and controlled mice were subjected to hindlimb ischemia (HLI) injury. The muscle ECs were sorted for single-cell RNA sequencing (scRNA-seq) analysis. HLI was also performed in high fat diet (HFD)-induced obese mice to examine the function of PPARδ in obese mice with delayed vascular repair. Adeno-associated virus type 1 (AAV1) carrying ICAM2 promoter to target endothelium for overexpressing PPARδ was injected into the injured muscles of normal chow- and HFD-fed obese mice before HLI surgery was performed. scRNA-seq analysis of ECs in ischemic muscles revealed a pivotal role of PPARδ in endothelial homeostasis. PPARδ expression was diminished both after HLI injury, and also in obese mice, which showed further delayed vascular repair. Endothelium-targeted delivery of PPARδ by AAV1 improved perfusion recovery, increased capillary density, restored endothelial integrity, suppressed vascular inflammation, and promoted muscle regeneration in ischemic hindlimbs of both lean and obese mice. Our study indicated the effectiveness of endothelium-targeted PPARδ overexpression for restoring functional vasculature after ischemic injury, which might be a promising option of gene therapy to treat PAD and CLI.

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.

A Ternary Synergistic eNOS Gene Delivery System Based on Calcium Ion and L-Arginine for Accelerating Angiogenesis by Maximizing NO Production.

PurposeThis study aimed to construct a delivery system based on L-arginine-modified calcium phosphate (CaP) to load eNOS plasmids (peNOS), which could amply nitric oxide (NO) to repair endothelial damage, promote angiogenic activities and alleviate inflammation.MethodspDNA-loaded CaP nanocomplex (CaP/pDNA) were prepared by co-precipitation method, subsequently modified by L-arginine. The gene transfection efficiency, pro-angiogenic and anti-inflammatory ability were investigated in vivo and in vitro. The therapeutic effect on ischemic hindlimb in vivo was assessed.ResultsL-arginine modification augmented the transfection efficiency of CaP/peNOS to elevate the eNOS expression, and then served as NO substrate catalyzed by eNOS. At the same time, calcium ions produced by degradation of CaP carriers enhanced the activity of eNOS. In vitro experiments, the loading capability and transfection performance of R(L)-CaP were confirmed to be superior to that of CaP. Additionally, HUVECs treated with R(L)-CaP/peNOS showed the strongest NO release, cell migration, tube formation and the lowest inflammatory levels compared to the CaP/peNOS and R(D)-CaP/peNOS groups. We also demonstrated the advantages of R(L)-CaP/peNOS in increasing blood reperfusion in hindlimb ischemia mice by accelerating angiogenesis and reducing inflammation, which can be attributed to the highest eNOS-derived NO production.ConclusionThe combination strategy of peNOS transfection, L-arginine supplement and calcium ions addition is a promising therapeutic approach for certain vascular diseases, based on the synergistic NO production.

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.

Quenching Epigenetic Drug Resistance Using Antihypoxic Microparticles in Glioblastoma Patient‐Derived Chips (Adv. Healthcare Mater. 8/2022)

Epigenetic Alterations In article number 2102226, Young Min Shin, Hak-Joon Sung, and co-workers demonstrate that loading anti-hypoxic microparticles onto glioblastoma (GBM) masses reduces epigenetic drug resistance and suppresses subsequent cell invasiveness. The results are verified using a 3D GBM chip with cell spheres in vitro under media perfusion and in vivo through chip implantation into mouse ischemic hind limbs.