Flow Recovery Research Articles

S100A9 deletion in microglia/macrophages ameliorates brain injury through the STAT6/PPARγ pathway in ischemic stroke.

Microglia and infiltrated macrophages (M/M) are integral components of the innate immune system that play a critical role in facilitating brain repair after ischemic stroke (IS) by clearing cell debris. Novel therapeutic strategies for IS therapy involve modulating M/M phenotype shifting. This study aims to elucidate the pivotal role of S100A9 in M/M and its downstream STAT6/PPARγ signaling pathway in neuroinflammation and phagocytosis after IS. In the clinical study, we initially detected the expression pattern of S100A9 in monocytes from patients with acute IS and investigated its association with the long-term prognosis. In the invivo study, we generated the S100A9 conditional knockout (CKO) mice and compared the stroke outcomes with the control group. We further tested the S100A9-specific inhibitor paqunimod (PQD), for its pharmaceutical effects on stroke outcomes. Transcriptomics and invitro studies were adopted to explore the mechanism of S100A9 in modulating the M/M phenotype, which involves the regulation of the STAT6/PPARγ signaling pathway. S100A9 was predominantly expressed in classical monocytes and was correlated with unfavorable outcomes in patients of IS. S100A9 CKO mitigated infarction volume and white matter injury, enhanced cerebral blood flow and functional recovery, and prompted anti-inflammation phenotype and efferocytosis after tMCAO. The STAT6/PPARγ pathway, an essential signaling cascade involved in immune response and inflammation, might be the downstream target mediated by S100A9 deletion, as evidenced by the STAT6 phosphorylation inhibitor AS1517499 abolishing the beneficial effect of S100A9 inhibition in tMCAO mice and cell lines. Moreover, S100A9 inhibition by PQD treatment protected against neuronal death invitro and brain injuries invivo. This study provides evidence for the first time that S100A9 in classical monocytes could potentially be a biomarker for predicting IS prognosis and reveals a novel therapeutic strategy for IS. By demonstrating that S100A9-mediated M/M polarization and phagocytosis can be reversed by S100A9 inhibition in a STAT6/PPARγ pathway-dependent manner, this study opens up new avenues for drug development in the field.

Effect of structure tool on nano-cutting surface integrity of single crystal γ-TiAl alloy via atomic simulation

To investigate the impact of microgroove geometry at the tool's flank face on the surface integrity of the workpiece, molecular dynamics simulation is utilized to establish a model of cutting single crystal γ-TiAl alloy using various structured nanodiamond tools. The effect of groove shape on the surface integrity of single crystal γ-TiAl alloys was investigated by analyzing mechanical properties, workpiece surface quality, and subsurface damage during the cutting process. The results show that non-structured tool cuts γ-TiAl alloy with the highest cutting force, and the V-groove tool cuts with the lowest friction coefficient, which is favorable for the removal of single crystal γ-TiAl alloy. Statistical atomic phase transitions during cutting confirmed that structured tools reduce subsurface damage, especially V-grooved tool reduce the generation of defective atoms. The circular arc grooved tool effectively reduces the subsurface temperature of the single crystal γ-TiAl alloy workpiece by reducing the friction at the tool chip interface due to lower normal cutting force. In addition, the circular arc grooved tool cutting has stronger atomic flow and surface recovery ability compared with other tools, and smaller surface roughness can be obtained. Therefore, selecting a reasonable structured tool can effectively improve the machinability of single crystal γ-TiAl alloy.

Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia

BackgroundCritical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal “cellular-cocktail” prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties.Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI.MethodsBalb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach.ResultsAT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified.ConclusionsThe combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

A KLF2-BMPER-Smad1/5 checkpoint regulates high fluid shear stress-mediated artery remodeling.

Vascular remodeling to match arterial diameter to tissue requirements commonly fails in ischemic disease. Endothelial cells sense fluid shear stress (FSS) from blood flow to maintain FSS within a narrow range in healthy vessels. Thus, high FSS induces vessel outward remodeling, but mechanisms are poorly understood. We previously reported that Smad1/5 is maximally activated at physiological FSS. Smad1/5 limits Akt activation, suggesting that inhibiting Smad1/5 may facilitate outward remodeling. Here we report that high FSS suppresses Smad1/5 by elevating KLF2, which induces the bone morphogenetic protein (BMP) pathway inhibitor, BMP-binding endothelial regulator (BMPER), thereby de-inhibiting Akt. In mice, surgically induced high FSS elevated BMPER expression, inactivated Smad1/5 and induced vessel outward remodeling. Endothelial BMPER deletion impaired blood flow recovery and vascular remodeling. Blocking endothelial cell Smad1/5 activation with BMP9/10 blocking antibodies improved vascular remodeling in mouse models of type 1 and type 2 diabetes. Suppression of Smad1/5 is thus a potential therapeutic approach for ischemic disease.

Time-domain diffuse correlation spectroscopy at large source detector separation for cerebral blood flow recovery.

Time-domain diffuse correlation spectroscopy (td-DCS) enables the depth discrimination in tissue's blood flow recovery, considering the fraction of photons detected with higher time of flight (TOF) and longer pathlength through the tissue. However, the recovery result depends on factors such as the instrument response function (IRF), analyzed TOF gate start time, gate width and the source-detector separation (SDS). In this research we evaluate the performance of the td-DCS technique at three SDSs of 1.5, 2 and 2.5 cm to recover cerebral blood flow (CBF). To do that we presented comprehensive characterization of the td-DCS system through a series of phantom experiments. First by quality metrices such as coefficient of variation and contrast-to-noise ratios, we identified optimal time gate(s) of the TOF to extract dynamics of particles. Then using sensitivity metrices, each SDS ability to detect dynamics of particles in superficial and deeper layer was evaluated. Finally, td-DCS at each SDS was tested on healthy volunteers during cuff occlusion test and breathing tasks. According to phantom measurements, the sensitivity to estimate perfusion within the deep layer located at depth of 1.5 cm from the surface can be increased more than two times when the SDS increases from 1.5 cm to 2.5 cm.

LncRNA H19/miR-107 regulates endothelial progenitor cell pyroptosis and promotes flow recovery of lower extremity ischemia through targeting FADD

BackgroundPeripheral artery disease (PAD) is an ischemic disease with a rising incidence worldwide. The lncRNA H19 (H19) is enriched in endothelial progenitor cells (EPCs), and transplantation of pyroptosis-resistant H19-overexpressed EPCs (oe-H19-EPCs) may promote vasculogenesis and blood flow recovery in PAD, especially with critical limb ischemia (CLI). MethodsEPCs isolated from human peripheral blood was characterized using immunofluorescence and flow cytometry. Cell proliferation was determined with CCK8 and EdU assays. Cell migration was assessed by Transwell and wound healing assays. The angiogenic potential was evaluated using tube formation assay. The pyroptosis pathway-related protein in EPCs was detected by western blot. The binding sites of H19 and FADD on miR-107 were analyzed using Luciferase assays. In vivo, oe-H19-EPCs were transplanted into a mouse ischemic limb model, and blood flow was detected by laser Doppler imaging. The transcriptional landscape behind the therapeutic effects of oe-H19-EPCs on ischemic limbs were examined with whole transcriptome sequencing. ResultsOverexpression of H19 in EPCs led to an increase in proliferation, migration, and tube formation abilities. These effects were mediated through pyroptosis pathway, which is regulated by the H19/miR-107/FADD axis. Transplantation of oe-H19-EPCs in a mouse ischemic limb model promoted vasculogenesis and blood flow recovery. Whole transcriptome sequencing indicated significant activation of vasculogenesis pathway in the ischemic limbs following treatment with oe-H19-EPCs. ConclusionsOverexpression of H19 increases FADD level by competitively binding to miR-107, leading to enhanced proliferation, migration, vasculogenesis, and inhibition of pyroptosis in EPCs. These effects ultimately promote the recovery of blood flow in CLI.

Assessment of cerebrovascular alterations induced by inflammatory response and oxidative–nitrative stress after traumatic intracranial hypertension and a potential mitigation strategy

The rapid perfusion of cerebral arteries leads to a significant increase in intracranial blood volume, exposing patients with traumatic brain injury to the risk of diffuse brain swelling or malignant brain herniation during decompressive craniectomy. The microcirculation and venous system are also involved in this process, but the precise mechanisms remain unclear. A physiological model of extremely high intracranial pressure was created in rats. This development triggered the TNF-α/NF-κB/iNOS axis in microglia, and released many inflammatory factors and reactive oxygen species/reactive nitrogen species, generating an excessive amount of peroxynitrite. Subsequently, the capillary wall cells especially pericytes exhibited severe degeneration and injury, the blood–brain barrier was disrupted, and a large number of blood cells were deposited within the microcirculation, resulting in a significant delay in the recovery of the microcirculation and venous blood flow compared to arterial flow, and this still persisted after decompressive craniectomy. Infliximab is a monoclonal antibody bound to TNF-α that effectively reduces the activity of TNF-α/NF-κB/iNOS axis. Treatment with Infliximab resulted in downregulation of inflammatory and oxidative–nitrative stress related factors, attenuation of capillary wall cells injury, and relative reduction of capillary hemostasis. These improved the delay in recovery of microcirculation and venous blood flow.

Validation of a Hybrid Domain Overlapping Coupling Between SAM and CFD Against the TALL-3D Transients

The system thermal-hydraulic (STH) code SAM has been coupled to the computational fluid dynamics (CFD) code Simcenter STARCCM+ utilizing a hybrid domain overlapping coupling method in space and an explicit coupling in time. The coupling aims to extend the STH code’s applicability to scenarios where local momentum and energy transfers are important yet difficult for STH standalone models to capture, such as three-dimensional (3D) mixing and thermal stratification. The coupling method’s numerical stability was previously verified against multiple test cases including two closed-loop configurations, and it was validated against a double T-junction experiment with 3D scalar mixing. In the present work, the method is validated against the TALL-3D STH/CFD coupling benchmark facility. TALL-3D is a liquid-metal facility with a large, pool-type enclosure (test section) that exhibits 3D flow effects to be modeled by a CFD code. The rest of the system exhibits approximately one-dimensional behavior that is well predicted by a STH code. First, a STAR-CCM+ CFD model of the 3D test section is validated against experimental temperature data. Then a SAM-STARCCM+ coupled model is validated against six different TALL-3D steady states and includes SAM standalone results for comparison. Last, the coupled model is validated against two TALL-3D transients: one exhibiting flow reversal in the test section and one exhibiting nonlinear, limit cycle oscillations (LCOs). For the first transient, the SAM-STARCCM+ coupled model properly predicts an increase in the test section’s inlet temperature during flow reversal, and this increase is not predicted by the SAM standalone model. The coupled model also better predicts initial flow recovery and subsequent oscillations as the system approaches a final natural circulation state. For the second transient, no true final steady state is observed due to LCOs. Neither the SAM-STARCCM+ coupled model nor the SAM standalone model can perfectly capture the experiment’s changing oscillation frequency during the transient. Nevertheless, the coupled model does reproduce the general oscillatory feedback observed. This is a significant achievement because the present coupling only uses an explicit coupling in time, as opposed to a tighter, semi-implicit coupling. In comparison, STH/CFD coupling efforts previously performed by other authors required a semi-implicit coupling to obtain similar results to this work. The strengths of the novel coupling scheme validated in the present paper lie in the superior convergence characteristics and the much simpler, nonintrusive implementation.

Synergistic effect of ex-vivo quality and quantity cultured mononuclear cells and mesenchymal stem cell therapy in ischemic hind limb model mice

IntroductionChronic limb-threatening ischemia (CLTI) is a condition characterized by peripheral arterial disease and tissue damage caused by reduced blood flow. New therapies using various cell types, such as mesenchymal stem cells (MSCs) and mononuclear cells (MNCs), have been developed for the patients unresponsive to conventional therapies. MSCs are promising because of their ability to secrete growth factors essential for vascularization, whereas MNCs contain endothelial progenitor cells that are important for blood vessel formation. However, conventional methods for isolating these cells have limitations, especially in patients with diabetes with dysfunctional cells. To overcome this problem, a culture method called quality and quantity cultured peripheral blood MNCs (MNC-QQ) was developed to efficiently produce high-quality cells from small amounts of peripheral blood. Combining MSCs with MNC-QQs has been hypothesized to enhance therapeutic outcomes. This study aimed to examine the angiogenic efficacy of MSCs with MNC-QQs in models with severe lower limb ischemia. MethodsMNC-QQ was manufactured from the peripheral blood of healthy volunteers, while human bone marrow derived MSCs were purchased. To verify the effects of the MSC and MNC-QQs combination in angiogenesis, we conducted the HUVEC tube formation assay. For in vivo experiments, we created an ischemic limb model using BALB/c nude mice. Saline, MSCs alone, and a combination of MSCs and MNC-QQs were administered intramuscularly into the ischemic limbs. Blood flow was measured over time using laser doppler, and the ischemic limbs were harvested 21 days later for HE staining and immunostaining for histological assessment. ResultsIn-vitro studies demonstrated increased angiogenesis when MSCs were combined with MNC-QQs compared with MSCs alone. In vivo experiments using a mouse model of severe lower limb ischemia showed that combination therapy significantly improved blood flow recovery and limb salvage compared with MSCs alone or saline treatment. Histological analysis revealed enhanced vessel density, arteriogenesis, muscle regeneration, and reduced fibrosis in the MSC + MNC-QQ group compared with those in the saline group. Although the specific interactions between MSCs and MNC-QQs have not been fully elucidated, combined therapy leverages the benefits of both cell types, resulting in improved outcomes for vascular regeneration. ConclusionsThis study highlights the potential of the simultaneous transplantation of MSCs and MNC-QQs as a promising therapeutic approach for CLTI, offering sustained long-term benefits for patients.

Timing of capillary enlargement after adenoviral VEGF gene transfer does not support post-ischemic skeletal muscle recovery in hyperlipidemic mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Finish Academy (Clinical Researcher 324070 and Research Project 339560). Finnish Foundation for Cardiovascular Research Background Transient capillary enlargement has been shown to be essential for successful post-ischemic tissue recovery as it may facilitate the return of arterial driving pressure by supporting collateral maturation. It also leads to an intussusceptive increase in capillary density that supports efficient oxygen delivery and muscle regeneration. Hyperlipidemia has been shown to blunt post-ischemic capillary reactivity and hinder skeletal muscle recovery from ischemia. Therefore, it can be hypothesized that correction of capillary reactivity might improve post-ischemic muscle recovery under hyperlipidemia. Purpose The aim of this study was to test the ability of angiogenic VEGF gene therapy in improving post-ischemic muscle recovery under hyperlipidemia. Methods AdVEGF gene transfer was performed into the calf muscles of aged, hyperlipidemic LDLR-/-ApoB100/100 mice after acute ischemia induction. The effects of AdVEGF on capillary phenotype, tissue edema, restoration of blood flow parameters, microvascular hemoglobin oxygenation and tissue damage/regeneration were evaluated for up to 29 days post-procedure as compared to AdLacZ control using immuno- and histological analyses, magnetic resonance, contrast-enhanced ultrasound and photoacoustic imaging, respectively. Results AdVEGF gene therapy was able to promote capillary enlargement that led to recovery of arterial driving pressure in ischemic LDLR-/-ApoB100/100 muscles. However, capillary enlargement induced by AdVEGF appeared late, lasted up to 29 days and did not promote intussusception. Consequently, the negative effect of long-lasting enlargement, i.e., massive tissue edema, delayed blood flow recovery and aggravated ischemic tissue damage. Conclusion Proper time of induction and duration of capillary enlargement are crucial to yield a functional vascular response supporting post-ischemic tissue recovery.

Injury to the sinus nodal artery during radiofrequency ablation of atrial fibrillation and atrial tachycardia: its prevalence and treatment with percutaneous coronary intervention

Abstract Background/Introduction Injury to the sinus node artery (SNA) leading to SN dysfunction during radiofrequency catheter ablation (RFCA) of atrial Fibrillation (AF) and atrial tachycardia (AT) has been reported. Purpose The purpose of this study was to elucidate the prevalence and clinical course of SNA injuries during RFCA of AF/AT and examine the efficacy of the percutaneous coronary intervention (PCI) to restore SN function. Methods We retrospectively analyzed data from 6,906 consecutive patients (pts) who underwent RFCA of AF/AT (6,379 pts with AF ablation and 527 pts with AT ablation) in two institutions between January 2013 and December 2022. Results Among 6,906 pts, 9 (0.13%) pts developed SN dysfunction due to SNA injury during RFCA (8 with AF, 1 with AT). Acute SNA injury (total occlusion or severe narrowing of the SNA) was confirmed by coronary angiography (CAG) during the RFCA session in 8/9 pts. In the remaining patient, SNA injury was suspected based on the CT angiography. The culprit sites of the SNA injury were located at the septal site of the superior vena cava-right atrium junction in 4 pts (44%), on the left atrial appendage ridge in 3 (33%), on the roof of the left atrium adjacent to the right superior pulmonary vein in 1 (11%), and in the persistent left superior vena cava in 1 (11%). PCI to the occluded SNA was performed in 8 of the 9 pts: 1) coronary blood flow of the SNA was restored by a 0.014" guidewire advancement into the occluded artery using a micro-catheter followed by direct nitroprusside injection in 4 pts; 2) the direct balloon angioplasty using 0.75-1.5 mm diameter balloons was required to maintain the blood flow of SNA in 3 pts; and 3) CAG revealed spontaneous recovery of the blood flow of SNA in one pt. The remaining patient did not receive the CAG/PCI. All 8 pts who underwent the PCI resulted in restoration of normal sinus rhythm. During a median of 44 months follow-up, all 8 pts who received the PCI maintained normal sinus rhythm, while one patient without PCI demonstrated persistent SN dysfunction. Conclusions SNA injury during RFCA is a rare but critical complication. Prompt recognition by CAG and subsequent PCI effectively restores SN function in pts with unexpected SN dysfunction during RFCA procedures.

Histotripsy and Catheter-Directed Lytic: Efficacy in Highly Retracted Porcine Clots In Vitro

ObjectiveStandard treatment for deep vein thrombosis (DVT) involves catheter-directed anticoagulants or thrombolytics, but the chronic thrombi present in many DVT cases are often resistant to this therapy. Histotripsy has been found to be a promising adjuvant treatment, using the mechanical action of cavitating bubble clouds to enhance thrombolytic activity. The objective of this study was to determine if histotripsy enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis in highly retracted porcine clots in vitro in a flow model of occlusive DVT. MethodsHighly retracted porcine whole blood clots were treated for 1 h with either catheter-directed saline (negative control), rt-PA (lytic control), histotripsy, DEFINITY and histotripsy or the combination of rt-PA and histotripsy with or without DEFINITY. Five-cycle, 1.5 MHz histotripsy pulses with a peak negative pressure of 33.2 MPa and pulse repetition frequency of 40 Hz were applied along the clot. B-Mode and passive cavitation images were acquired during histotripsy insonation to monitor bubble activity. ResultsClots subjected to histotripsy with and without rt-PA exhibited greater thrombolytic efficacy than controls (7.0% flow recovery or lower), and histotripsy with rt-PA was more efficacious than histotripsy with saline (86.1 ± 10.2% compared with 61.7 ± 19.8% flow recovery). The addition of DEFINITY to histotripsy with or without rt-PA did not enhance either thrombolytic efficacy or cavitation dose. Cavitation dose generally did not correlate with thrombolytic efficacy. ConclusionEnhancement of thrombolytic efficacy was achieved using histotripsy, with and without catheter-directed rt-PA, in the presence of physiologic flow. This suggests these treatments may be effective as therapy for DVT.

MiR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation

The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3′UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B.

Electroacupuncture enhances cerebral blood perfusion by inhibiting HIF-1α in rat subarachnoid hemorrhage

ObjectiveCerebral blood perfusion (CBP) reduction is a prevalent complication following subarachnoid hemorrhage (SAH) in clinical practice, often associated with long-term cognitive impairment and prognosis. Electroacupuncture (EA), a widely utilized traditional Chinese therapy for central nervous system disorders, has demonstrated promising therapeutic effects. This study aims to investigate the therapeutic potential of EA in restoring CBP in SAH rats and to explore the mechanisms involving HIF-1α in this process. MethodsRats were randomly assigned to one of five groups, including Sham, SAH, EA, EA + Saline, and EA + dimethyloxallyl glycine (DMOG) groups. EA treatment was administered for 10 min daily, while DMOG were intraperitoneally injected. Behavioral tests, cerebral blood flow monitoring, vascular thickness measurement, western blotting, and immunofluorescence staining were conducted to assess the therapeutic effects of EA on cerebral blood flow. ResultsSAH resulted in elevated levels of HIF-1α, endothelin (ET), ICAM-1, P-SELECTIN, E-SELECTIN, and decreased level of eNOS in the brain. This led to cerebral vasospasm, decreased CBF, and cognitive deficits in the rat SAH model. EA intervention downregulated the expression of HIF-1α, ET, ICAM-1, P-SELECTIN, and E-SELECTIN, while increasing eNOS expression. This alleviated cerebral vasospasm, restored CBF, and improved cognitive function. However, the administration of the HIF-1α stabilizer (DMOG) counteracted the therapeutic effects of EA. ConclusionEA promotes the recovery of cerebral blood flow after SAH injury, attenuates cerebral vasospasm, and accelerates the recovery of cognitive dysfunction, and its mechanism of action may be related to the inhibition of the HIF-1α signaling pathway.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

Abstract 2030: Endothelial Deletion Of Bag3 Does Not Impair Ischemic Limb Recovery In A Murine Model Of Peripheral Artery Disease

Background: Peripheral artery disease (PAD) is a highly prevalent vascular occlusive disease which mostly affects the lower limb. Emerging studies proved the critical functions of endothelial cells in arteriogenesis and ischemic limb blood flow recovery. However, very few targets with endothelial cell exclusivity have been identified. Here, we explored the role of endothelial BCL-2 associated athanogene 3 (Bag3) in ischemic limb recovery. Hypothesis: We hypothesized that the loss of endothelial specific BAG3 would attenuate blood flow and muscle recovery following HLI. Methods: Hind limb ischemia (HLI) was induced in Bag3 Cdh5(PAC)-CreERT2 male and female mice after tamoxifen (2mg/day x 5 days) or vehicle treatment ( n ≥12/group). Limb blood flow was measured by laser Doppler perfusion imaging (LDPI) and isometric muscle force production, vascular density and muscle morphology were assessed after HLI. Results: LDPI analysis revealed no differences in the blood flow between groups at HLI d7 ( n=9 ; day 7, 21.86% ischemic/non-ischemic ± 3.80% vs 18.05% ± 1.96% SEM; p=0.05). There was no deficit in isometric force recovery of the EDL ( n ≥12; 57.76 ± 15.92 vs 83.05 ± 27.12 SEM; p =0.05) in KO animals at HLI d7. Similar rates of CD31+ vessel disorganization was observed in both groups at HLI d7, ( n =7; 4626.0 um 2 CD31+ area ± 869 vs 4870.9 ± 1056 SEM) consistent with recovering ischemic tissue. There were also no differences in blood flow recovery assessed by LDPI at HLI d28 ( n =7; 50.74% ischemic/non-ischemic ± 7.4% vs 59.17 ± 8.21; n=9). Conclusions: Endothelial cell specific Bag3 knockout did not impair vascular morphology or perfusion recovery at baseline or following HLI in a murine model of peripheral artery disease. No differences in muscle morphology or contractile function at baseline or after HLI at either timepoint were observed. In summary, we reveal an unexpectedly minor role of endothelial cell BAG3 in tissue recovery following ischemic injury.

Abstract 3026: Genetic Deletion Of Skeletal Muscle Bag3 Exacerbates Ischemic Myopathy

Background: Bcl2-associated athanogene 3 (BAG3) is a multifunctional adaptor protein that regulates diverse cellular functions. Mutations in BAG3 are causally linked to myofibrillar myopathy and dilated cardiomyopathy in humans and now appear to be one of the most common to cause human disease. We previously identified a murine coding variant in Bag3 that contributes to hindlimb ischemia (HLI) pathology, however, no cell specific data on Bag3’s role in PAD pathology exists. Hypothesis: We hypothesized that muscle specific BAG3 is required for both the restoration of blood flow and muscle recovery following HLI. Methods: We generated a genetic model of lifelong muscle cell (mature fiber and progenitor) Bag3 deletion (Pax7-Cre; Bag3 -/- ) on a BL6 parental background. Male and Female mice (12-20 weeks; WT: n=45; KO: n=32) were evaluated at baseline and HLI d7 and d28 for blood flow via LDPI, isometric muscle force production, vascular density and muscle morphology. Bag3 deletion was validated by recombination, qRT-PCR and Western Blotting in whole tissue, isolated myofibers, and FACS muscle progenitor cells. Results: KO reduced muscle force production (n=18; 145.8mN Force ± 28.6 vs 56.9 ± 16.8 SEM; p=0.0208) and capillary perfusion (n=7; 0.17 CD31+/Lectin+ vessels ± 0.02 vs 0.09 ± 0.01 SEM p=0.0491) indicative of the multi-compartmental effect of BAG3 loss in skeletal muscle at HLI d7. Extended HLI (d28) revealed a KO phenotype characterized by reduced blood flow recovery (LDPI; n= 7; .68 ischemic/non limb ± 0.5 vs .38 ± 0.6 SEM p=0.0011) and sustained myopathy including reduced myofiber size ( n =7; 1585um 2 ± 161 vs 1093 ± 169 SEM; p =0.05) and increased tissue lesion size ( n =7; 2.5% muscle area ± 2.0 vs 48.3% ± 21.6; p =0.03). Conclusion: Loss of skeletal muscle Bag3 exacerbates myopathy and reduces capillary perfusion and blood flow recovery following HLI and demonstrate a requirement for BAG3 in recovery from limb ischemia.

Abstract 1142: Effect Of Chronic E-cigarette Nicotine Exposure On A Mouse Model Of Hind-limb Ischemia

Background: The rise of e-cigarette use in the United States has been rapid, yet its impact on peripheral artery disease (PAD) remains largely unexplored. This study investigates the relationship between e-cigarette use and PAD using a mouse ischemic hindlimb model. Method: Mice aged 6-8 weeks were divided into four groups. Three groups inhaled e-cigarette vapor continuously for 14, 20, or 28 days prior, and for 28 days after hindlimb ischemia (HLI) surgery, using an InExpose system (Figure 1). A control group was exposed to room air (RA) before and after HLI. Laser-Doppler perfusion imaging tracked hindlimb blood flow, and functional deficits were assessed using the modified ischemia scale. Figure 1: Methodology Results: Blood perfusion in the ischemic hindlimb of the e-cigarette groups (EC-14, EC-20, EC-28) was significantly lower than the non-vaping group at 21 days post-surgery. In the EC-14 group, perfusion improved after 10 days of vaping cessation (Figure 2). Additionally, there was a reduction in platelet and leukocyte counts in the e-cigarette groups compared to RA, with no difference in red blood cells. Necrosis and claw damage were observed in EC-20 and EC-28 groups but not in non-vaping mice. Figure 2: Representative images of perfusion recovery measured by LDPI. Conclusion: Chronic e-cigarette nicotine exposure impairs blood flow recovery and causes necrosis in mice with hind limb ischemia, potentially due to perturbed angiogenesis and arteriogenesis. This study highlights the detrimental effects of e-cigarette nicotine on vascular health.

Abstract 3122: The Role Of Oct4 In Endothelial Cell Sexual Dimorphism And Angiogenesis

Background: Biological sex differences play a critical role in vascular diseases. Using endothelial ( EC ) lineage tracing atherosclerotic mice and cultured mouse and human aortic EC, we recently found that female EC have lower angiogenic potential, higher expression of pro-inflammatory genes and intracellular ROS, and worse mitochondria functions than male EC. However, the mechanisms responsible for this EC sexual dimorphism are still unknown. We previously identified the pluripotency factor OCT4 as a critical regulator of EC characteristics, including angiogenic potential, ROS accumulation, and mitochondrial functions. Therefore, this study aimed to test whether OCT4 is responsible for sex-dependent differences in EC in vivo and in vitro . Methods: We used male and female EC-specific tamoxifen-inducible OCT4 knockout and EC-lineage tracing (EC-OCT4 KO) mice to study the role of OCT4 in non-atherosclerotic conditions leading to pro-inflammatory angiogenesis, such as hind-limb ischemia and skin injury models. Male and female mouse aortic EC (MAEC) were isolated from EC-OCT4 KO and wild-type littermate mice for in vitro experiments. We performed BrdU assay, EC tube formation in Matrigel (in vitro angiogenesis), wound scratch assay, and angiogenic proteome profile array to evaluate angiogenic potential. Results: Compared to wild-type mice, EC-OCT4 KO male mice exhibited accelerated blood flow recovery and wound healing after hind-limb ischemia and skin injury. However, EC-OCT4 KO female mice showed no difference in blood flow recovery and delayed skin wound healing compared to control mice. In vitro , although male and female OCT4-KO MAEC had higher angiogenic potential than control, the phenotype in female EC was less pronounced. Mechanistically, we found that male OCT4 KO MAEC express higher levels of monocyte cytokine protein 1 ( MCP1 ). Furthermore, the inhibition of MCP1 in male OCT4 KO MAEC, but not in female cells, led to significant decreases in cell migration, proliferation, and endothelial tube formation in Matrigel. Conclusions: Our data indicate that OCT4-dependent mechanisms contribute to the sex differences observed in EC, suggesting a novel biological role for pluripotency signaling in vascular pathologies and homeostasis.

Diversity Outbred Mouse Model Recapitulates Human Heterogeneity in the Development of Western Diet-Induced Complications

Background: Diet-induced obesity (DIO) increases risk for cardiovascular disease largely due to compromised gut barrier integrity and systemic inflammation. Animal models for understanding the pathophysiology of complications associated with DIO do not take address the genetic factors, which determine the individual variation in the susceptibility to develop complications. Hypothesis: DO mice recapitulate the heterogeneity of western diet-induced metabolic syndrome, gut barrier disruption and systemic inflammation. Methods: DO mice were fed with either control (CD) or western diet (WD) (Teklad) (n = 10) for 16 weeks. Blood glucose levels, glucose tolerance and the intestinal permeability were determined. Inflammatory cells in the bone marrow or blood were characterized in basal conditions or in response to hindlimb ischemia (HLI). Blood flow recovery following HLI was measured by using Laser Doppler Imaging system and the intestinal-stem-cells (ISCs) were characterized by using markers, Lgr5, Olfm4 and Wnt3. Results: Wide variation in the weight gain, hyperglycemia and glucose tolerance in DO mice was observed following WD (n = 10). Similarly, wide variation was observed in Wnt3 expression and in the density of ISCs in colon of DO mice on WD. Heterogeneity in the ISC layer correlated with the variation in the intestinal permeability induced by WD compared to the CD ( p < 0.05, n = 10). The number of inflammatory cells (monocytes – CD45+Ly6G−Ly6C+CD115+, neutrophils - CD45+Ly6G+Ly6C+CD115−, pro-inflammatory macrophages - CD45+CD11b+Ly6G−Ly6C+F4/80+CCR2+CD80+ and anti-inflammatory macrophages - CD45+CD11b+Ly6G−Ly6C+F4/80+CX3CR1+CD206+) in the BM or blood showed heterogeneity in WD-DO mice in accordance with variability in the glucose tolerance. Accordingly, blood flow recovery following HLI showed wide variation with three mice on WD showing full recovery, which correlated with variability in the mobilization of inflammatory cells (n = 9). Conclusion: DO mouse model depicts human diversity in response to WD-induced changes in gut barrier integrity, inflammation and ischemic vascular repair therefore serves as a suitable model for evaluating pharmacological agents that would be effective in a diverse population. Jackson Laboratory Diversity Outbred Pilot Award National Institute of Aging (AG056881). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.