Perfusion Recovery Research Articles

The impact of type 2 diabetes on short term myocardial morphological, functional, and energetic recovery post coronary artery bypass surgery in patients with ischemic heart disease

Abstract Background Type 2 diabetes (T2D) confers three times higher mortality in patients with ischemic heart disease (IHD). Moreover, amongst patients with IHD those with diabetes were shown to have higher long-term mortality from heart failure despite angiographic or surgical revascularisation with coronary artery bypass grafting (CABG). Impaired myocardial structural, functional, and energetic recovery may underpin this epidemiological observation. Consequently, using cardiovascular magnetic resonance and phosphorus-magnetic resonance spectroscopy scans, we aimed to assess the effect of T2D comorbidity on myocardial structural, functional, perfusion, and energetic recovery post-CABG in patients with IHD. Methods Seventy-seven IHD patients with (n=39) and without T2D (n=38) awaiting CABG were recruited. One-month pre- and 6-months post-CABG, cardiac phosphocreatine to ATP ratio (PCr/ATP), global longitudinal shortening (GLS) and 6-minute walk-distance (6MWD) were assessed in the study cohorts. The rest and adenosine-stress myocardial blood flow (MBF) assessments were only performed 6-months post-CABG. Results Pre-CABG, IHD-T2D patients had higher LV concentricity (LV mass to LV end diastolic volume ratio) (IHD-T2D:0.84[0.74,0.93],IHD-noT2D:0.72[0.67,0.77]g/mL,P=0.03]. worse PCr/ATP T2D (IHD-T2D:1.5[1.4,1.7],IHD-noT2D:1.8[1.6, 2.0];P=0.03) and shorter 6-minute walk distance (IHD-T2D:338[307,370],IHD-noT2D:399[364,434]m,P=0.007). There were no significant differences in LV volumes, ejection fraction or GLS between the two groups pre-CABG. Post-CABG, there was no longer a significant difference in cardiac energetics or 6-minute walk distances between the two groups due to a significant improvement in PCr/ATP in the IHD-T2D group. LV concentricity remained significantly elevated in the IHD-T2D group post CABG (IHD-T2D:0.82[0.75,0.88],IHD-noT2D:0.73[0.68,0.79]g/mL,P=0.04). Neither group showed a significant change in LVEF or GLS from the pre-CABG values, and post-CABG there were no differences in the rest or stress myocardial blood flows between the two groups. Conclusions Six months after CABG, neither IHD patients with T2D, nor IHD patients without T2D show any significant changes in LV structural remodelling or in contractile function. T2D does not jeopardise the short-term recovery in myocardial energetics or exercise distance post-CABG, with IHD patients with T2D showing more pronounced improvements post-CABG in both these assessments than patients without T2D. Larger longitudinal studies are needed to better understand the mechanisms of adverse longer-term cardiovascular outcomes suggested by epidemiological studies in IHD patients with T2D.

Hypoperfused myocardium and systolic function in patients with AMI undergoing complete revascularization

Abstract Background and Aim Coronary revascularization is the standard treatment for acute myocardial infarction (AMI), while the restoration of epicardial flow is non-parallel to myocardial reperfusion. This study sought to investigate the dynamic of myocardial perfusion and its association with left ventricular (LV) systolic function in AMI patients undergoing complete revascularization. Methods AMI patients who underwent complete revascularization were prospectively enrolled, and hybrid positron emission tomography/magnetic resonance (PET/MR) was performed at 2 months and 1 year. Hypoperfused myocardium was defined as myocardium with reduced perfusion and normal metabolism. Results Forty-five patients (59.80 ± 10.68 years, 80.0% male) were enrolled, and hypoperfused myocardium (median: 12% of LV) was detected in 21 (46.7%) patients 2 months post-AMI. Patients with hypoperfused myocardium (Group A) had significantly higher hypersensitive C-reactive protein (hs-CRP) (p = 0.016) and lower T2 value (p = 0.010) than those without (Group B). In the follow-up PET/MR at 1 year, significant LV ejection fraction improvement was observed in Group A (56.39 ± 9.76% vs. 54.58 ± 10.39%, p = 0.016) but not in Group B (51.63 ± 13.09% vs. 50.79 ± 16.95%, p = 0.615). Six (31.6%) patients in Group A showed recovery of perfusion, of which there were more females (p = 0.025), fewer diabetics (p = 0.044), or overweight (p = 0.035), with better global longitudinal strain (GLS) (p = 0.005). Conclusions Myocardial hypoperfusion is not rare and evolves over time in AMI patients undergoing complete revascularization, of which the reversal is probably associated with improved LV systolic function.Structured Graphical AbstractRepresentative PET/MR image of 2 separat

Synthetic Antiangiogenic Vascular Endothelial Growth Factor-A Splice Variant Revascularizes Ischemic Muscle in Peripheral Artery Disease.

Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF165a and antiangiogenic VEGF165b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF165a) to SLTRKD (in VEGF165b). We have recently shown that VEGF165b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF165b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear. Hypoxia serum starvation was used as an invitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF165b. A synthetic anti-angiogenic VEGF165b splice variant in which the KD residues were switched to RR (VEGF165bKD→RR) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity invitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF165a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF165bKD→RR over VEGF165a is due to its ability to induce P38-activation in ischemic endothelial cells. Our data shows that the KD residues regulate VEGF165b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF165bKD→RR isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.

Microcirculatory Perfusion Disturbances During Veno-Arterial Extracorporeal Membrane Oxygenation: ASystematic Review.

Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used in case of potentially reversible cardiac failure and restores systemic hemodynamics. However, whether this is followed by improvement of microcirculatory perfusion is unknown. Moreover, critically ill patients have possible pre-existing microcirculatory perfusion disturbances. Therefore, this review provides an overview of alterations in sublingual microcirculatory perfusion in critically ill adult patients receiving VA-ECMO support. Pubmed, Embase (Ovid), Cochrane Central Register of Controlled Trials, and Web of Science were systematically searched according to PRISMA guidelines. Studies reporting sublingual microcirculatory perfusion measurements in adult patients supported by VA-ECMO were included. Outcome parameters included small vessel density (SVD), perfused vessel density (PVD), perfused small vessel density (PSVD), proportion of perfused vessels (PPV), microvascular flow index (MFI) and the heterogeneity index (HI). The protocol was registered at PROSPERO (CRD42021243930). The search identified 1215 studies of which 11 were included. Cardiogenic shock was the most common indication for VA-ECMO (n=8). Three studies report increased PSVD, PPV, and MFI 24 hours after initiation of ECMO compared to pre-ECMO. Nonetheless, microcirculatory perfusion stabilized thereafter. Four out of four studies showed higher PSVD and PPV in survivors compared to non-survivors. Over time, survivors showed recovery of microcirculatory perfusion within hours of initiation of ECMO, whereas this was absent in non-survivors. Notwithstanding the limited sample, VA-ECMO seems to improve microcirculatory perfusion shortly after initiation of ECMO, especially in survivors. Further research in larger cohorts is needed to clarify the longitudinal effects of ECMO on microcirculatory perfusion.

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.

An unexpected role of IL10 in mesoderm induction and differentiation from pluripotent stem cells: Implications in zebrafish angiogenic sprouting, vascular organoid development, and therapeutic angiogenesis

Mesoderm induction is a crucial step for vascular cell specification, vascular development and vasculogenesis. However, the cellular and molecular mechanisms underlying mesoderm induction remain elusive. In the present study, a chemically-defined differentiation protocol was used to induce mesoderm formation and generate functional vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs). Zebrafish larvae were used to detect an in vivo function of interleukin 10 (IL10) in mesoderm formation and vascular development. A three dimensional approach was used to create hiPSC-derived blood vessel organoid (BVO) and explore a potential impact of IL10 on BVO formation. A murine model hind limb ischemia was applied to investigate a therapeutic potential of hiPSC-derived cells treated with or without IL10 during differentiation. We found that IL10 was significantly and specifically up-regulated during mesoderm stage of vascular differentiation. IL10 addition in mesoderm induction media dramatically increased mesoderm induction and vascular cell generation from hiPSCs, whereas an opposite effect was observed with IL10 inhibition. Mechanistic studies revealed that IL10 promotes mesoderm formation and vascular cell differentiation by activating signal transducer and activator of transcription 3 signal pathway. Functional studies with an in vivo model system confirmed that knockdown of IL10 using morpholino antisense oligonucleotides in zebrafish larvae caused defective mesoderm formation, angiogenic sprouting and vascular development. Additionally, our data also show IL10 promotes blood vessel organoid development and enhances vasculogenesis and angiogenesis. Importantly, we demonstrate that IL10 treatment during mesoderm induction stage enhances blood flow perfusion recovery and increases vasculogenesis and therapeutic angiogenesis after hind limb ischemia. Our data, therefore, demonstrate a regulatory role for IL10 in mesoderm formation from hiPSCs and during zebrafish vascular development, providing novel insights into mesoderm induction and vascular cell specifications.

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.

“All in one” lipid-polymer nanodelivery system for gene therapy of ischemic diseases

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Allogenic Vertebral Body Adherent Mesenchymal Stromal Cells Promote Muscle Recovery in Diabetic Mouse Model of Limb Ischemia

Chronic limb threatening ischemia (CLTI) carries a significant risk for amputation especially in diabetic patients with poor options for revascularization. Phase I trials have demonstrated efficacy of allogeneic mesenchymal stromal cells (MSC) in treating diabetic CLTI. Vertebral bone adherent mesenchymal stromal cells (vBA-MSC) are derived from vertebral bodies of deceased organ donors which offer the distinct advantage of providing a 1,000x greater yield compared to that of living donor bone aspiration. This study describes the effects of intramuscular injection of allogenic vBA-MSC in promoting limb perfusion and muscle recovery in a diabetic CLTI mouse model. A CLTI mouse model was created through unilateral ligation of the femoral artery in male polygenic diabetic TALLYHO mice. Treated mice were injected with vBA-MSC into the gracilis muscle of the ischemic limb 7 days post ligation. Gastrocnemius or tibialis muscle was assessed post-mortem for fibrosis by collagen staining, capillary density via immunohistochemistry, and mRNA by quantitative real time PCR. Laser Doppler perfusion imaging and plantar flexion muscle testing were performed to quantify changes in limb perfusion and muscle function. Compared to vehicle control, treated mice demonstrated indicators of muscle recovery including decreased fibrosis, increased perfusion, muscle torque, and angiogenesis. PCR analysis of muscle obtained 7- and 30-days post vBA-MSC injection showed an upregulation in expression of MyoD1 (p = 0.03) and MyH3 (p = 0.008) mRNA representing muscle regeneration, VEGF-A (p = 0.002 ; p = 0.004) signifying angiogenesis as well as IL-10 (p < 0.001), T regulatory cell marker Foxp3 (p = 0.04), and M2-biased macrophage marker Mrc1 (CD206) (p = 0.02). These findings indicate human allogeneic vBA-MSC ameliorate ischemic muscle damage and rescue muscle function. These results in a murine model will enable further studies to develop potential therapies for diabetic CLTI patients.

The Complement System Is Essential for Arteriogenesis by Enhancing Sterile Inflammation as a Relevant Step in Collateral Artery Growth.

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 -/-) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 -/- mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 -/- mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 -/- mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68+) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68+/MRC1-) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 -/- mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

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.

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.

Power-Doppler Ultrasonic Imaging of Peripheral Perfusion in Diabetic Mice.

We explored the capabilities of power-Doppler ultrasonic (PD-US) imaging without contrast enhancement for monitoring changes in muscle perfusion over time. Ischemic recovery was observed in healthy and type II diabetic male and female mice with and without exercise. In separate studies, perfusion was measured during and after 5-min ischemic periods and during four-week recovery periods following irreversible femoral ligation. A goal was to assess how well PD-US estimates tracked the diabetic-related changes in endothelial function that influenced perfusion. The average perfusion recovery time following femoral ligation increased 47% in diabetic males and 74% in diabetic females compared with non-diabetic mice. Flow-mediated dilation in conduit arteries and the reactive hyperemia index in resistive vessels each declined by one half in sedentary diabetic mice compared with sedentary non-diabetic mice. We found that exercise reduced the loss of endothelial function from diabetes in both sexes. The reproducibility of perfusion measurements was limited primarily by our ability to select the same region in muscle and to effectively filter tissue clutter. PD-US measurements can precisely follow site-specific changes in skeletal muscle perfusion related to diabetes over time, which fills the need for techniques capable of regularly monitoring atherosclerotic changes leading to ischemic vascular pathologies.

The Inhibition of γ-Aminobutyric Acid B1 Receptor Regulates Angiogenesis via the Hippo/YAP Signaling Pathway

Promoting the establishment of collateral circulation is essential for chronic lower extremity ischemia. However, no effective therapeutic drugs have yet been developed. Recent studies discovered that in the peripheral arteries, there are GABAB1 receptors expressed in endothelial cells and smooth muscle cells, these receptors may have some effects in regulating vascular functions, but the precise mechanism is not yet clear. This study explores the effect of GABAB1 receptor inhibition on angiogenesis and its regulatory mechanism. The expression of GABAB1 in HUVECs was knocked down using shRNA transfection, and effects in HUVECs' proliferation, migration, and tube formation ability were detected. Western blot and RT-PCR were used to verify the signal pathway. The murine hind limb ischemia model was used to verify the effect of CGP35348, an antagonist of GABAB1R, on the recovery of blood flow perfusion and angiogenesis in ischemic tissues. Cell proliferation, migration, and tube formation ability were improved after GABAB1 receptor knockdown in HUVECs. The phosphorylation of the HIPPO/YAP pathway decreased, while the effect of promoting angiogenesis increased. After treating the ischemic hindlimbs of mice with GABAB1 receptor antagonists, the blood flow perfusion recovered, and the angiogenesis increased. These findings demonstrate the effect of GABAB1 receptor inhibition on the HIPPO/YAP pathway in regulating angiogenesis, suggesting that inhibiting GABAB1 receptor levels might be a novel approach for chronic lower extremity ischemia diseases.

Unveiling impaired vascular function and cellular heterogeneity in diabetic donor-derived vascular organoids.

Vascular organoids (VOs), derived from induced pluripotent stem cells (iPSCs), hold promise as in vitro disease models and drug screening platforms. However, their ability to faithfully recapitulate human vascular disease and cellular composition remains unclear. In this study, we demonstrate that VOs derived from iPSCs of donors with diabetes (DB-VOs) exhibit impaired vascular function compared to non-diabetic VOs (ND-VOs). DB-VOs display elevated levels of reactive oxygen species (ROS), heightened mitochondrial content and activity, increased proinflammatory cytokines, and reduced blood perfusion recovery in vivo. Through comprehensive single-cell RNA sequencing, we uncover molecular and functional differences, as well as signaling networks, between vascular cell types and clusters within DB-VOs. Our analysis identifies major vascular cell types (endothelial cells [ECs], pericytes, and vascular smooth muscle cells) within VOs, highlighting the dichotomy between ECs and mural cells. We also demonstrate the potential need for additional inductions using organ-specific differentiation factors to promote organ-specific identity in VOs. Furthermore, we observe basal heterogeneity within VOs and significant differences between DB-VOs and ND-VOs. Notably, we identify a subpopulation of ECs specific to DB-VOs, showing overrepresentation in the ROS pathway and underrepresentation in the angiogenesis hallmark, indicating signs of aberrant angiogenesis in diabetes. Our findings underscore the potential of VOs for modeling diabetic vasculopathy, emphasize the importance of investigating cellular heterogeneity within VOs for disease modeling and drug discovery, and provide evidence of GAP43 (neuromodulin) expression in ECs, particularly in DB-VOs, with implications for vascular development and disease.

Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies. Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies. Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed. Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.

A suction blister model to characterize epidermal wound healing and evaluate the efficacy of the topical wound healing agent INM-755 in healthy volunteers.

Non-healing wounds represent a substantial medical burden with few effective treatments available. To address this challenge, we developed a novel epidermal wound healing model using suction blisters in healthy volunteers. This model allowed for the comprehensive assessment of wound healing dynamics and the evaluation of INM-755, a topical cream containing cannabinol, as a potential therapeutic agent. Two clinical studies were conducted: an observational study and an interventional study. In both studies, healthy volunteers underwent a suction blister procedure on their lower back, creating open epidermal wounds. Wound healing parameters were assessed using advanced imaging systems. Skin barrier function and perfusion were evaluated through trans epidermal water loss (TEWL) and dynamic optical coherence tomography (D-OCT), respectively. The observational study demonstrated the successful and reproducible induction of blisters and the removal of epidermal sheet, enabling quantifiable measurements of wound healing parameters over time. Re-epithelialization was observed, revealing recovery of skin barrier function and perfusion. In the interventional study, differences of treatments over time were quantified using the above-described techniques. Despite differences from disease-specific blistering, our developed model provides a valuable platform for studying wound healing mechanisms and assessing novel therapeutic interventions. The sensitivity to treatment effects demonstrated in our study underscores the potential utility of this model in early-phase clinical drug development programs targeting wound healing disorders.

THROMBOLYSIS IN ST- ELEVATION MYOCARDIALINFRACTION PATIENTS: CLINICAL PREDICTORS OF REPERFUSIONAND ANGIOGRAPHIC OUTCOMES

Background: Thrombolysis is an alternative strategy for reperfusion in the management of ST- Elevation Myocardial Infraction (STEMI) patients, when percutaneous coronary intervention (PCI) cannot be performed. However, using clinical markers of reperfusion, thrombolysis is not always successful for all patients and immediate angiographicrescue PCI is indicated. Aim of the Work: Thegoal of this clinical study was to determinethe angiographic efficacy of thrombolysis in STEMI patients. Besides its correlation in successful or failed thrombolysis (FT) based on clinicalcriteria of reperfusion. Material/Methods: The study population included patients with acute STEMI within 12 hours of onset chest pain, who were admitted to The Cardiac Intensive Care Unit of cardiology department, atUniversityHospital Center Mohammed VI of Marrakech Morocco, receiving thrombolysis with Tenecteplase, during the period from January2021 to January2023. Repeated Electrocardiogram (EKG) at baseline, 90 minutes after Thrombolysis to define FT and PCIwereperformedfor all patients. Results:121 patients with a mean age of 56 years were recruited in the study. Successful thrombolysis was present in 60% of patients (n = 73) in Group I, while 40% of patients (n = 48) had FT in Group II. There was no significant difference between patients in both groups regarding gender, risk factors for coronary artery disease like dyslipidemia, diabetes mellitus, obesity, smoking, and myocardial infraction territory on the EKG. However, hypertension, delayed presentation after six hours of onset chest pain, and severe alteration of the systolic ejection fraction were mostly present in Group II. The TIMI III flow rates represent 76%(n = 93) of the patients in the study, with higher rates in both groups, at 80% versus 70% for groups I and II, respectively. Conclusion: The study confirmed that unsuccessful thrombolysisdoes not mean always-unsuccessful restoring of epicardial coronary flow.As a result, our current results clearly demonstrate the benefits of fibrinolysisfor STEMI patients when resources are limited for primary PCI. However, Additional clinical studies are also needed to investigate the recovery of microvascular and tissular perfusion to improve myocardial function and long-term clinical outcomes.

CCL11 released by GSDMD-mediated macrophage pyroptosis regulates angiogenesis after hindlimb ischemia

Peripheral vascular disease (PVD) is an emerging public health burden with a high rate of disability and mortality. Gasdermin D (GSDMD) has been reported to exert pyroptosis and play a critical role in the pathophysiology of many cardiovascular diseases. We ought to determine the role of GSDMD in the regulation of perfusion recovery after hindlimb ischemia (HLI). Our study revealed that GSDMD-mediated pyroptosis occurred in HLI. GSDMD deletion aggravated perfusion recovery and angiogenesis in vitro and in vivo. However, how GSDMD regulates angiogenesis after ischemic injury remains unclear. We then found that GSDMD-mediated pyroptosis exerted the angiogenic capacity in macrophages rather than endothelial cells after HLI. GSDMD deletion led to a lower level of CCL11 in mice serum. GSDMD knockdown in macrophages downregulated the expression and decreased the releasing level of CCL11. Furthermore, recombinant CCL11 improved endothelial functions and angiogenesis, which was attenuated by CCL11 antibody. Taken together, these results demonstrate that GSDMD promotes angiogenesis by releasing CCL11, thereby improving blood flow perfusion recovery after hindlimb ischemic injury. Therefore, CCL11 may be a novel target for prevention and treatment of vascular ischemic diseases.

Noninvasive Real-Time Assessment of Nipple-Areola Complex Perfusion Using Laser Speckle Contrast Imaging in Direct-to-Implant Breast Reconstruction.

Nipple-areola complex (NAC) necrosis is a major complication for breast reconstruction after nipple-sparing mastectomy. Although intraoperative indocyanine green angiography helps to assess the viability of tissue, the imaging could be conservative which may lead to aggressive resection. The plastic surgeons are eager to know the perfusion changes of NAC throughout the perioperative period. In this prospective cohort study, the authors enrolled patients who underwent NSM and immediate direct-to-implant breast reconstruction. All patients underwent laser speckle contrast imaging before surgery, immediately after mastectomy, after implant placement, and 24h and 72h after surgery. A total of 94 breasts were analyzed, including 64 breasts healed with viable NAC and 30 breasts with NAC necrosis. In viable NACs, the average blood supply decreased to 56% after NSM and 42% after reconstruction, then recovered to 68% and 80% at 24-h and 72-h post-operation. In necrotic NACs, the average blood supply decreased to 33% after NSM and 24% after reconstruction, and partial perfusion recovery was also recorded at 24-h (31%) and 72-h (37%) post-operation. The cutoff value for predicting NAC viability is 40% after NSM and 25% after implant placement. The study quantified the NAC perfusion changes during the perioperative period. NAC perfusion decreased significantly after NSM and would be the lowest after the end of breast reconstruction. Viable NACs displayed more perfusion during the operation and showed significant nipple revascularization after breast reconstruction. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .