Hindlimb Ischemia Research Articles

What Is the Best Experimental Model for Developing Novel Therapeutics in Peripheral Artery Disease?

More than 200 million people worldwide have peripheral artery disease (PAD). PAD affects the quality of life and is associated with significant morbidity and mortality. Standard treatment for severe cases of PAD is surgical or endovascular revascularization. However, up to 30% of patients are not candidates for open or endovascular procedures, due to high operative risk or unfavorable vascular involvement. Furthermore, revascularization procedures may be insufficient to adequately improve microvascular tissue perfusion, wound healing, or limb salvage. Accordingly, regardless of advances in treatment modalities, outcomes of patients with PAD have remained unfavorable. Therefore, new medical therapeutic approaches are much needed. Small animal models are indispensable tools for the understanding of PAD physiopathology and the development of novel medical therapies. Development of animal models that more closely mimic the pathophysiology (with occlusive atherothrombosis and chronic development of limb ischemia) can incorporate the cardiovascular risk factors associated with this disease state, and focus on more clinically relevant outcomes is critical. In practice, this means using both animals that develop atherosclerosis and methods for the application of gradual arterial occlusion to induce hind limb ischemia. Doing so will likely help identify novel targets for intervention and overcome some principal challenges confronted by previous clinical trials. While various rodent models are discussed, the optimal animal model is yet to be defined.

Targeting the histone methyltransferase SETD7 rescues diabetes-induced impairment of angiogenic response by transcriptional repression of semaphorin 3G

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

Microvascular blood flow disturbances predict poor outcome of revascularization in CLTI patients

Abstract Background We have recently established hypoxic microvascular remodelling as a critical pathomechanistic factor that limits oxygen diffusion to muscle in chronic limb threatening ischaemia (CLTI). Using animal models of experimental hind limb ischaemia we have also displayed the biological relevance of post-ischaemic microvascular remodelling in skeletal muscle. Our results indicate that differential microvascular changes associate to muscle damage and repair. The natural microvascular dynamics that inherently promote muscle recovery are defected in CLTI patients. Particularly, a progressive arterialization of capillaries in CLTI muscle differentiates the dynamics of the microvasculature from initially assisting muscle regeneration to aggravating ischaemic damage. Methods To establish the clinical relevance of the post-ischaemic microvascular changes in CLTI, we now have studied the effect of microvascular remodelling on the outcome of CLTI patients undergoing surgical or endovascular revascularization. Contrast enhanced ultrasound was used preoperatively to determine the microvascular status of CLTI patients (n=36) scheduled for immediate revascularization. Outcome events such as: hospitalization; reoperation; leg amputation; or death, were postoperatively followed from medical records up to 6 months after revascularization. Patients were grouped according to muscle capillary transit time (Tct) being either similar (n=18) or shorter (n=18) to that in the contralateral legs of the patients, and treatment outcomes were compared between groups. Results The results display consistent increases in postoperative event rates in all measured outcomes in the ShortTct group as compared to controls (at 1 month 8 vs. 2 events (rate ratio 4.00 (95%CI: 0.85-18.84, p=0.080)); at 3 months 13 vs. 4 events (rate ratio 3.25 (95%CI: 1.06-9.97, p=0.039)); at 6 months 17 vs. 9 events (rate ratio 1.89 (95%CI: 0.84-4.24, p=0.123))). For example, 5 vs. 0; 7 vs. 1; and 7 vs. 4 reoperations were detected in the ShortTct group as compared to controls at 1, 3 and 6 months, respectively. By 6 months after initial revascularization also 2 deaths were recorded in the ShortTct group as compared to no deaths in the controls. Conclusions Despite still small samples size in this study, these results suggest that microvascular blood flow disturbances, such as short preoperative muscle capillary transit time, can predict poor outcome of CLTI patients after revascularization. Importantly, the results also identify patients with microvascular blood flow disturbances a subgroup of CLTI patients that should likely receive more specialized care to improve outcomes. The results also call for immediate actions from the scientific community to improve clinical detection of microvascular alterations and to develop strategies to normalize microvascular function in CLTI patients.

Inhibition of microRNA-181c-5p rescues diabetes-impaired angiogenesis through activation of key mediators of angiogenesis, cell survival and novel genes, elmo3 and trib1

Abstract Introduction Patients with diabetes have impaired angiogenesis and poor coronary collateral vessel formation post-myocardial infarction (MI), which associates with higher mortality. There is a significant unmet clinical need for new agents that stimulate angiogenesis in response to ischaemia in diabetes. We have identified that miR-181c-5p has anti-angiogenic properties, but it’s role in diabetes remains unknown. Aim To elucidate the role of miR-181c-5p in diabetes-impaired angiogenesis. Methods Human coronary artery endothelial cells were transfected with a miR-181c-5p inhibitor (antimiR-181c-5p) or negative control (antimiR-Neg), exposed to glucose (5–25mM, 48h) then underwent Matrigel tubulogenesis assay or Boyden Chamber migration assay. Levels of proteins important for angiogenesis (e.g., VEGFA, p-ERK2, p-eNOS) were determined by Western Blot. Whole transcriptome sequencing was performed in vitro to identify novel gene targets of miR-181c-5p. In vivo, diabetic mice underwent hindlimb ischaemia or wound healing surgery and were injected with antimiR-181c-5p or antimiR-Neg. Tissues were extracted early (day 3) and late (days 10-14) post-surgery. Hindlimb blood-flow reperfusion was measured by Laser Doppler imaging. Hindlimb apoptosis was assessed by TUNEL and necrotic area was assessed by H&E. Wound area was calculated daily. Neovascularisation was assessed by CD31 (capillaries) and α-actin (arterioles) immunostaining. Results Inhibition of miR-181c-5p increased endothelial tubule number (+28%, P<0.01), tubule length (+12%, P<0.01) and cell migration (+67%, P<0.05). This associated with increased VEGFA (+21%, P<0.05) and p-ERK2 (+32%, P<0.05). Whole transcriptome and pathway analysis revealed changes to angiogenesis pathways and identified a first-time involvement of genes Elmo3 and Trib1 in the pro-angiogenic action of antimiR-181c-5p in diabetes. In vivo, inhibition of miR-181c-5p increased blood flow reperfusion to ischaemic hindlimbs (+30%, P<0.001) and arteriolar density (+45%, P<0.05) in diabetic mice. Mechanistically, this was associated with early changes to mediators of angiogenesis, Erk2 mRNA (+35%, P<0.05), p-ERK2 (+35%, P<0.05) and Trib1 mRNA (+80%, P<0.05); cell survival, Bcl-2 mRNA (+44%, P<0.05); and late apoptotic clearance, Elmo3 mRNA (+57%, P<0.001). Furthermore, this was also associated with an increase in late stage hindlimb apoptosis (+94%, P<0.05) and reduced necrotic area (-90%, P<0.05) in diabetic mice. Inhibition of miR-181c-5p increased diabetic wound closure (+22%, P<0.01), wound capillaries (+61%, P<0.05), Bcl-2 mRNA (+52%, P<0.05) and Elmo3 mRNA (+50%, P<0.05) in diabetic wounds. Conclusions Inhibition of miR-181c-5p rescues diabetes-impaired angiogenesis by activation of angiogenesis and cell survival mediators, and through novel genes, Trib1 and Elmo3. Our findings have implications for a novel miRNA-based strategy that improves myocardial neovascularisation and the prognosis of diabetic patients post-MI.

In-situ engineering of native extracellular matrix to improve vascularization and tissue regeneration at the ischemic injury site

Ischemic injury causes dynamic damage to the native extracellular matrix (ECM), which plays a key role in tissue homeostasis and regeneration by providing structural support, facilitating force transmission, and transducing key signals to cells. The main approach aimed at repairing injury to ischemic tissues is restoration of vascular function. Due to their potential to form capillary niches, endothelial cells (ECs) are of greatest interest for vascular regeneration. Integrin binding to ECM is crucial for cell anchorage to the surrounding matrix, spreading, migration, and further activation of intracellular signaling pathways. In this study, we proposed to establish an in-situ engineering strategy to remodel the ECM at the ischemic site to guide EC endogenous binding and establish effective EC/ECM interactions to promote revascularization. We designed and constructed a dual-function molecule (LXW7)2-SILY, which is comprised of two functional domains: the first one (LXW7) binds to integrin αvβ3 expressed on ECs, and the second one (SILY) binds to collagen. In vitro, we confirmed (LXW7)2-SILY improved EC adhesion and survival. After in situ injection, (LXW7)2-SILY showed stable retention at the injured area and promoted revascularization, blood perfusion, and tissue regeneration in a mouse hindlimb ischemia model.Graphical

Synergistic effect of Hypoxic Conditioning and Cell-Tethering Colloidal Gels enhanced Productivity of MSC Paracrine Factors and Accelerated Vessel Regeneration.

Microporous hydrogels have been widely used for delivering therapeutic cells. However, several critical issues, such as the lack of control over the harsh environment they are subjected to under pathological conditions and rapid egression of cells from the hydrogels, have produced limited therapeutic outcomes. To address these critical challenges, cell-tethering and hypoxic conditioning colloidal hydrogels containing mesenchymal stem cells (MSCs) are introduced to increase the productivity of paracrine factors locally and in a long-term manner. Cell-tethering colloidal hydrogels that are composed of tyramine-conjugated gelatin prevent cells from egressing through on-cell oxidative phenolic crosslinks while providing mechanical stimulation and interconnected microporous networks to allow for host-implant interactions. Oxygenating microparticlesencapsulated in tyramine-conjugated colloidal microgels continuously generated oxygen for 2 weeks with rapid diffusion, resulting in maintaining a mild hypoxic condition while MSCs consumed oxygen under severe hypoxia. Synergistically, local retention of MSCs within the mild hypoxic-conditioned and mechanically robust colloidal hydrogels significantly increased the secretion of various angiogenic cytokines and chemokines. The oxygenating colloidal hydrogels induced anti-inflammatory responses, reduced cellular apoptosis, and promoted numerous large blood vessels in vivo. Finally, mice injected with the MSC-tethered oxygenating colloidal hydrogels significantly improved blood flow restoration and muscle regeneration in a hindlimb ischemia (HLI) model.

Feasibility of Monitoring Tissue Properties During Microcirculation Disorder Using a Compact Fiber-Based Probe With Sapphire Tip.

One of the urgent tasks of modern medicine is to detect microcirculation disorder during surgery to avoid possible consequences like tissue hypoxia, ischemia, and necrosis. To address this issue, in this article, we propose a compact probe with sapphire tip and optical sensing based on the principle of spatially resolved diffuse reflectance analysis. It allows for intraoperative measurement of tissue effective attenuation coefficient and its alteration during the changes of tissue condition, caused by microcirculation disorder. The results of experimental studies using (1) a tissue-mimicking phantom based on lipid emulsion and hemoglobin and (2) a model of hindlimb ischemia performed in a rat demonstrated the ability to detect rapid changes of tissue attenuation confirming the feasibility of the probe to sense the stressful exposure. Due to a compact design of the probe, it could be useful for rather wide surgical operations and diagnostic purposes as an auxiliary instrument.

Contrast-Enhanced Ultrasound of Mouse Models of Hindlimb Ischemia Reveals Persistent Perfusion Deficits and Distinctive Muscle Perfusion Patterns

ObjectiveMouse models of hindlimb ischemia (HLI) are used to study peripheral arterial disease and evaluate novel therapies. Contrast-enhanced ultrasound (CEUS) is a noninvasive perfusion measurement technique that is increasingly being employed in these models. The objective of this study was to evaluate two models of severe HLI by CEUS to characterize perfusion recovery and muscle perfusion patterns. MethodsMice undergoing double femoral artery ligation were measured by CEUS and laser Doppler perfusion imaging (LDPI) at baseline and 1–150 d postsurgery. A second group undergoing femoral artery ligation and excision was measured 1–28 d postsurgery. ResultsBy LDPI, both surgeries showed robust perfusion recovery by 14 d postsurgery. However, by CEUS only a ∼40% perfusion recovery plateau was reached in either group. These results are consistent with our previous work, employing a less severe single femoral artery ligation, that showed perfusion in the ischemic limb does not return to normal by 150 d postsurgery. Cluster analysis of muscle perfusion patterns indicated 3–5 different patterns at day 1 postsurgery. The double ligation model yielded significantly less variable perfusion patterns, suggesting that it can provide more reproducible results. ConclusionContrary to LDPI, perfusion as measured by CEUS never fully recovers after hindlimb surgery, even when followed 28–150 d postsurgery. Individual mice can manifest different patterns of muscle perfusion to the same surgery, but these patterns are conserved within and between different surgical techniques. These results may have significant implications for the evaluation of novel therapeutics to treat PAD in mice.

The Systemic Effect of Ischemia Training and Its Impact on Bone Marrow-Derived Monocytes.

Monocytes are innate immune cells that play a central role in inflammation, an essential component during neovascularization. Our recent publication demonstrated that ischemia training by 24 h unilateral occlusion of the femoral artery (FA) can modify bone marrow-derived monocytes (BM-Mono), allowing them to improve collateral remodeling in a mouse model of hindlimb ischemia. Here, we expand on our previous findings, investigating a potential systemic effect of ischemia training and how this training can impact BM-Mono. BM-Mono from mice exposed to ischemia training (24 h) or Sham (same surgical procedure without femoral artery occlusion-ischemia training) procedures were used as donors in adoptive transfer experiments where recipients were subjected to hindlimb ischemia. Donor cells were divided corresponding to the limb from which they were isolated (left-limb previously subjected to 24 h ischemia and right-contralateral limb). Recipients who received 24 h ischemic-trained monocytes isolated from either limb had remarkable blood flow recovery compared to recipients with Sham monocytes (monocytes isolated from Sham group-no ischemia training). Since these data suggested a systemic effect of ischemic training, circulating extracellular vesicles (EVs) were investigated as potential players. EVs were isolated from both groups, 24 h-trained and Sham, and the former showed increased expression of histone deacetylase 1 (HDAC1), which is known to downregulate 24-dehydrocholesterol reductase (Dhcr24) gene expression. Since we previously revealed that ischemia training downregulates Dhcr24 in BM-Mono, we incubated EVs from 24 h-trained and Sham groups with wild-type (WT) BM-Mono and demonstrated that WT BM-Mono incubated with 24 h-trained EVs had lower gene expression of Dhcr24 and an HDAC1 inhibitor blunted this effect. Next, we repeated the adoptive transfer experiment using Dhcr24 KO mice as donors of BM-Mono for WT mice subjected to hindlimb ischemia. Recipients who received Dhcr24 KO BM-Mono had greater limb perfusion than those who received WT BM-Mono. Further, we focused on the 24 h-trained monocytes (which previously showed downregulation of Dhcr24 gene expression and higher desmosterol) to test the expression of a few genes downstream of the desmosterol pathway, confirm the Dhcr24 protein level and assess its differentiation in M2-like macrophage phenotype. We found that 24 h-trained BM-Mono had greater expression of key genes in the desmosterol pathway, such as liver X receptors (LXRs) and ATP-binding cassette transporter (ABCA1), and we confirmed low protein expression of Dhcr24. Further, we demonstrated that ischemic-trained BM-Mono polarized towards an anti-inflammatory M2 macrophage phenotype. Finally, we demonstrated that 24 h-trained monocytes adhere less to endothelial cells, and the same pattern was shown by WT BM-Mono treated with Dhcr24 inhibitor. Ischemia training leads to a systemic effect that, at least in part, involves circulating EVs and potential epigenetic modification in BM-Mono. These ischemic-trained BM-Mono demonstrated an anti-inflammatory phenotype towards M2 macrophage differentiation and less ability to adhere to endothelial cells, which is associated with the downregulation of Dhcr24 in those cells. These data together suggest that Dhcr24 might be an important target within monocytes to improve the outcomes of hindlimb ischemia.

Glucosamine-Mediated Hexosamine Biosynthesis Pathway Activation Uses ATF4 to Promote "Exercise-Like" Angiogenesis and Perfusion Recovery in PAD.

Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway. Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS-/-) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF165a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care. On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS-/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF165a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group). In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.

Insulin-like growth factor-2 mRNA-binding protein 2 facilitates post-ischemic angiogenesis by increasing the stability of fibroblast growth factor 2 mRNA and its protein expression

BackgroundPost-ischemic angiogenesis is crucial for reestablishing blood flow in conditions such as peripheral artery disease (PAD). The role of insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP2) in post-transcriptional RNA metabolism and its involvement in post-ischemic angiogenesis remains unclear. MethodsUsing a human GEO database and a hind-limb ischemia (HLI) mouse model, the predominant isoform IGF2BP2 in ischemic gastrocnemius tissue was identified. Adeno-associated virus with the Tie1 promoter induced IGF2BP2 overexpression in the HLI model, evaluating the expression of vascular structural proteins (CD31 and α-SMA) and blood flow recovery after HLI. In vitro experiments with human umbilical vein endothelial cells (HUVECs) demonstrated that lentivirus-mediated IGF2BP2 overexpression upregulates cell proliferation, migration, and tube formation. GeneCards, RNAct databases, and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) predicted IGF2BP2 interactions with fibroblast growth factor 2 (FGF2) mRNA, and actinomycin D treatment, binding site predictions and CLIP-seq data further confirmed this interaction. Furthermore, western blotting, enzyme-linked immunosorbent assay, and RNA immunoprecipitation followed by RT-qPCR were performed to validate IGF2BP2's interaction with FGF2 mRNA and to assess its role in stabilizing FGF2 mRNA, as well as its impact on FGF2 protein expression. ResultsHLI reduced IGF2BP2 expression in the gastrocnemius tissue, which gradually increased during blood flow recovery. IGF2BP2 overexpression in HLI mice accelerated blood flow recovery and increased capillary and small artery densities. The overexpression of IGF2BP2 in HUVECs stimulated proliferation, migration, and tube formation by interacting with FGF2 mRNA to increase its stability. This interaction resulted in increased levels of FGF2 protein and secretion, ultimately promoting angiogenesis. ConclusionsIGF2BP2 contributes to blood flow restoration post-ischemia in vivo and promotes angiogenesis in HUVECs by enhancing FGF2 mRNA stability and FGF2 protein expression and secretion. These findings underscore IGF2BP2's therapeutic potential in ischemic conditions, such as PAD.

Regulation of angiogenesis by signal sequence-derived peptides.

The neuropilin-like, Discoidin, CUB and LCCL domain containing 2 (DCBLD2) is a transmembrane protein with an unusually long signal sequence (SS) composed of N-terminal (N) and C-terminal (C) subdomains, separated by a transition (tra) subdomain. DCBLD2 interacts with VEGFR-2 and regulates VEGF-induced endothelial cell signaling, proliferation and migration, as well as angiogenesis. The exact mechanisms by which DCBLD2 interacts with VEGFR2 to modulate VEGF signaling remain unclear. Searching for VEGFR2 interacting DCBLD2 domains, we generated various constructs containing different DCBLD2 domain combinations and conducted co-immunoprecipitation and signaling studies in HEK 293T and endothelial cells. Several peptides were synthesized based on the identified domain, and their effect on VEGF signaling was assessed in vitro in cell culture and in vivo using matrigel plug and corneal micropocket assays. The effect of the lead peptide was further evaluated using a murine hindlimb ischemia model. DCBLD2 SS interacted with VEGFR2 and promoted VEGF signaling. SS was not cleaved in the mature DCBLD2 and its hydrophobic transmembrane 'traC' segment, but not the 'N' subdomain, was involved in DCBLD2-VEGFR2 interaction. The smallest unit in DCBLD2 SS that interacts with VEGFR2 was the L5VL5 sequence. Even after the central valine was removed, the L10 sequence mimicked the DCBLD2 SS traC's effect on VEGF-signaling, while shorter or longer poly-leucine sequences were less effective. Finally, a synthetic traC peptide enhanced VEGF signaling in vitro, promoted VEGF-induced angiogenesis in vivo, and improved blood flow recovery following hindlimb ischemia. DCBLD2 SS along with its derivative peptides can promote VEGFR2 signaling and angiogenesis. Synthetic peptides based on DCBLD2 SS hold promise as therapeutic agents for regulating angiogenesis. Importantly these findings refine the traditional view of signal sequences as mere targeting elements, revealing a role in cellular signaling. This opens new avenues for research and therapeutic strategies.

Integrated single-cell RNA-seq analysis reveals mitochondrial calcium signaling as a modulator of endothelial-to-mesenchymal transition.

Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.

Attenuation of esophageal anastomotic stricture through remote ischemic conditioning in a rat model

Anastomotic stricture is a typical complication of esophageal atresia surgery. Remote ischemic conditioning (RIC) has demonstrated multiorgan benefits, however, its efficacy in the esophagus remains unclear. This study aimed to investigate whether applying RIC after esophageal resection and anastomosis in rats could attenuate esophageal stricture and improve inflammation. Sixty-five male Sprague–Dawley rats were categorized into the following groups: controls with no surgery, resection and anastomosis only, resection and anastomosis with RIC once, and resection and anastomosis with RIC twice. RIC included three cycles of hind-limb ischemia followed by reperfusion. Inflammatory markers associated with the interleukin 6/Janus kinase/ signal transducer and activator of transcription 3 (IL-6/JAK/STAT3) and tumor necrosis factor-alpha/nuclear factor-κB (TNF-α/NF-kB) signaling pathways were evaluated with RNA and protein works. The RIC groups showed significantly lower stricture rates, lower inflammatory markers levels than the resection and anastomosis-only group. The RIC groups had significantly lower IL-6 and TNFa levels than the resection and anastomosis-only group, confirming the inhibitory role of remote ischemic conditioning in the IL-6/JAK/STAT3 and TNF-α/NF-kB signaling pathways. RIC after esophageal resection and anastomosis can reduce the inflammatory response, improving strictures at the esophageal anastomosis site, to be a novel noninvasive intervention for reducing esophageal anastomotic strictures.

Effects of L-Ornithine-L-Aspartate on Angiogenesis and Perfusion in Subacute Hind Limb Ischemia: Preliminary Study.

The current treatment options for peripheral arterial disease (PAD) are limited due to a lack of significant high-level evidence to inform clinical decisions and unfavorable outcomes in terms of cost-effectiveness and amputation rates. In order to suggest the use of the commercially available L-Ornithine-L-Aspartate (LOLA) for treating PAD, we induced hind limb ischemia (HLI) by unilaterally ligating the femoral artery in a rat model. The rats were randomly divided into three groups, with seven rats assigned to each group: group 1 (control), group 2 (sorbitol), and group 3 (LOLA). Intraperitoneal injections were administered five times on post-operative days (PODs) 3, 5, 7, 10, and 12. Perfusion imaging was conducted on PODs 7 and 14 and compared to pre-operative perfusion imaging. Immunohistochemistry staining and Western blotting were performed after the final perfusion imaging. Group 3 showed a significant increase in perfusion, high CD31-positive capillary lumen density, and substantial overexpression of VEGF in the ischemic limb during the subacute phase of HLI. In conclusion, this study provides the first documented evidence of angiogenesis and perfusion recovery in the subacute phase of the HLI model following the administration of LOLA. With LOLA readily available on the commercial market, the implementation of LOLA treatment for PAD in humans can be expedited compared to other therapies still in the developmental stage.

Abstract We139: 3D Chromatin Architectures and Transcription Regulation in Diabetic Endothelial Dysfunction

Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC high-glu ). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC high-glu and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC high-glu and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC high-glu . We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC high-glu significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.

Abstract We111: 3D spheroids composed by induced Skeletal Muscle Progenitor Cells and Mesenchymal Stem Cells derived from human Pluripotent Stem Cells can recapitulate embryonic niches in hindlimb ischemia model

Introduction: Peripheral artery disease (PAD) is a significant comorbidity in aging population regarding diminished quality of life and increased mortality rates. There are urgent needs for developing advanced therapeutics for functional limb salvage beyond conventional revascularization. We hypothesized that 3D spheroids composed by induced Skeletal Muscle Progenitor Cells (iSKMPs) and Mesenchymal Stem Cells (iMSCs) could recapitulate embryonic niches and provide promising regenerative potentials in hindlimb ischemia (HLI) models. Methods: We manufactured scaffold-free, self-aggregated 3D spheroids using microcavity plates with ultra-low attachment surface. 3D spheroids were designed into 3 groups according to the induced cell types derived from Human Pluripotent Stem Cells (hPSCs); 1) iSKMPs only, 2) iMSCs only, 3) iSKMPs + iMSCs mixed. We transplanted 3D spheroids into the ischemic area and compared their therapeutic potentials using Laser Doppler Perfusion Imaging (LDPI) and immunohistochemical staining analysis. Results: iSKMPs secreted various angiogenic factors including IGF, HGF and FGF and facilitated tube formation and migration of endothelial cells (ECs). iSKMPs also showed the differentiation potential into myofibers in defined 2D culture condition. During the 28-day follow-up period, the transplantation of 3D mixed spheroids not only increased blood perfusion in ischemia limb, but also significantly reduced amputation rate compared with those of control and other 3D single spheroid groups. Notably, in vivo capacity of muscle regeneration was more potent in 3D mixed spheroid group with higher rate of engraftment, myotube formation, and vascularization. These findings suggested that iSKMPs played a significant role in limb salvage via myogenesis and angiogenesis, and their cell-to-cell interaction with iMSCs within 3D spheroids provided a favorable microenvironment to enhance the engraftment and maturation of iSKMPs in ischemic hindlimb. Conclusion: We demonstrated that the transplantation of 3D spheroids composed by iSKMPs and iMSCs could recapitulate developmental embryonic niches and provide potent regenerative potentials in HLI models. These findings provide compelling evidence that this injectable platform of 3D mixed spheroids can be a promising tool to rescue limb amputation in patients with PAD.

Abstract Tu023: The VEGF-A splice variants as a mechanism of impaired Inflammatory Angiogenesis in the context of advanced aging.

Background and Objectives: Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairment of angiogenesis responses to injury. Critical ischemic limb and wound injury animal models have shown impact of monocytes/macrophages as a major source of angiogenic mediators leading to new arterial growth. Aging has also been associated with impairment of blood flow recovery and reduced VEGF-A expression in animals. However, recombinant VEGF-A administration or clinical trials involving VEGF-A have not fully overcome the foot perfusion and prevent limb/leg amputation. We sought to understand molecular mechanisms of reduced VEGF-A expression which remain incomplete in the context of advanced aging. Results: Firstly, in hindlimb ischemia mouse model, our data from liposomes containing-clodronate treated mice (12-weeks C57Bl6 with macrophage depletion model ) ( Fig. 1a ) demonstrate impairment of blood flow recovery compared to Control group. On day 3 post-surgery, VEGF-A and VEGF-A isoforms expression (VEGF-A 165 a and VEGF-A 165 b, respectively) were reduced in ischemic muscle. Fluorescence staining showed a reduction of macrophages (CD68 + ) was also associated with reduced endothelial cells (CD31 + ). Using a myeloid-specific and inducible deleted VEGF fl/fl mice, we then have shown a key role of early inflammatory macrophages stage as a major source of VEGF-A required for angiogenesis in young mice (Fig. 1b). Secondly, bone marrow-derived-macrophages (BMDMs) from C57Bl6 104-weeks mice also demonstrate reduced VEGF-A expression. Aged BMDMs demonstrate reduced VEGF-A 165 a while VEGF-A 165 b was highly increased. Polarized BMDMs from 12-weeks demonstrate increased VEGFR1 in “M2" macrophages while VEGFR2 was increased in “M1” macrophages. On the other hand, BMDMs from 104-weeks demonstrate reduced VEGFR2 expression while VEGFR1 was unaffected compared to young. Lastly, 104-weeks mice also demonstrate severe impairment of blood flow recovery ( Fig. 2 ) after surgery and reduced VEGF-A and VEGF-A 165 a in ischemic muscle tissue. VEGF-A 165 b was increased and both receptors, VEGF-R1/R2 levels ( Fig. 3 ) were also reduced. Fluorescence staining on day 3 post ligation demonstrates reduced endothelial cells recruitment in muscle tissue while macrophage (CD68+) number was similar. Conclusion: Our investigations will contribute to define mechanisms whereby vascular injury is associated with VEGF-A isoforms specific switch in the context of advanced aging.

Abstract We012: Sheet like extracellular matrix secreting single cell microvesicles for reprogrammed endothelial cell therapy towards vascular regeneration

To enhance cell survival and engraftment for cell therapy, cell encapsulated injectable biomaterials have been widely used. However, the challenges were hypoxia and nutrient shortage in the core, inflammatory cell infiltration and limited distribution of cells from injection sites. Encapsulation of single cells in sub-100-micron microgels with higher surface to volume ratios provides more efficient oxygen and mass exchange. However, typical production of single-cell-laden microgels by microfluidics devices results in cell damage and compromised biological properties. We hypothesized that stimuli responsive amphiphilic copolymer bearing RGD peptides may self-assemble around single cells induced by cell adhesion moieties would result in single cell microvesicles (MV). A stimuli responsive amphiphilic copolymer GPM (Gelatin Poly glycerol sebacate Methacrylate) was synthesized by grafting hydrophobic synthetic polymer PGS into hydrophilic natural polymer gelatin via methacrylation followed by aza-Michael addition reaction. Single cell microvesicles of endothelial cells (EC-MVs) were fabricated by self-assembly of GPM at room temperature. For in vivo studies, sub-50-micron EC-MVs made of reprogrammed endothelial cells (rECs) were clustered into 200 micron spheres (mGPM) using sodium alginate and injected into a hindlimb ischemia model to investigate cell survival and biological function. The mGPM microspheres functioned as a barrier from immune cell infiltration and also enhanced cellular distribution. The encapsulated rECs were released over 2-4 wks in vivo and were shown robust engraftment, migration, and survival in limb tissues, promoting blood flow recovery and sustained vascular regeneration over 6 months. Interestingly, in in vitro culture, rEC-MVs secreted sheet like extra cellular matrix (ECM) which appeared to make an important role for vessel formation (neovascularization) in vivo (Figure 1). The current study clearly demonstrated the favorable biological applicability of GPM single cell MVs toward vascular regeneration by reprogrammed ECs.

Glycolytic PFKFB3 and Glycogenic UGP2 Axis Regulates Perfusion Recovery in Experimental Hind Limb Ischemia.

Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle. Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model. Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis). Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.