Vascular Recovery Research Articles

Retinal vascular recovery revealed by retinal imaging following neonatal hypoxia ischemia in mice: Is there a role for tyrosine kinase receptor modulation?

ObjectiveHypoxic ischemic encephalopathy (HIE) secondary to perinatal asphyxia leads to long-term visual disabilities. Dilated retinal exams in human newborns with HIE is an emerging diagnostic tool, but phenotypes of hypoxia ischemia (HI) related retinal vascular injury are unclear. 7,8-Dihydroxyflavone (7,8-DHF) is a TrkB agonist with protective effects on HI-related brain damage. We studied retinal vessels in a mouse model of neonatal HIE and the efficacy of 7,8-DHF in ameliorating HI-related retinal vascular injury. MethodsC57BL6/J mice at post-natal day (P) 9 received unilateral left carotid artery ligation followed by exposure to 10 % oxygen for 50 min. Phosphate buffered saline or 7,8-DHF (5 mg/kg) were administered daily for 7 days intraperitoneally. Control groups of naïve or carotid artery ligation only mice were studied. Fluorescein angiography was performed in acute (two weeks post-exposure) and chronic (four weeks post-exposure) time points. Retinal artery width, retinal vein width, and collateral vessel length were quantified. ResultsLigation of the common carotid artery alone caused retinal artery dilation in acute and chronic time points, but had no effect on retinal veins. At acute time point, HI caused increased retinal artery vasodilation, but was reversed by 7,8-DHF. HI caused short collateral vessel formation in ipsilateral eyes, rescued by 7,8-DHF treatment. ConclusionRetinal artery vasodilation and collateral vessel formation due to HI were rescued by 7,8-DHF treatment. Retinal and collateral vessel monitoring could be diagnostic biomarkers for HI severity. Studies to elucidate mechanisms of 7,8-DHF action on retinal vessels could aid development of therapies for neonatal HI.

Development and characterization of a preclinical total marrow irradiation conditioning-based bone marrow transplant model for sickle cell disease

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2–4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.

Abstract P2023: Non-cardiomyocytes From Chronic Unloaded Post LVAD Heart Form Organized Complex Neovascular Structures When Compared To Those From Heart Failure.

Introduction: Chronic unloading of the left ventricle (LV) has shown increased endothelial density suggestive of a vascular recovery. Mechanisms of such neovascularization have not been fully explored. Hypothesis: Non-myocyte transitions may play a role in neovascularization in cardiac recovery. Methods: We utilized fresh cardiac tissue obtained from patients at the time of LVAD (pre-LVAD core - representing HF) and from a LVAD patient at the time of transplant (Post-LVAD-representing chronic unloaded heart). Using disaggregation, cardiac non-myocytes were isolated, and the resulting pellet of cardiac non-myocytes was divided and plated in both fibroblast (FGM) and endothelial (EGM) growth media. Results: Cells from post-LVAD patient in EGM (grown on glass coverslip) showed a complex organizational network with the presence of CD31+ cells in vessel like structures surrounded by vimentin positive cells (See Figure). These networks were not observed in any of the other culture conditions. We also observed that cells from post-LVAD grew much faster compared to pre-LVAD cells. Conclusions: To our knowledge, this is the first-time disaggregated cells from human heart samples have been studied in culture. These preliminary observations support the ability of non-cardiomyocytes in a chronic unloaded and rested heart to form neovascular structures not seen in cells from the diseased state of heart failure. This platform can serve to further understand the behavior and role of complex non-myocyte interactions in cardiac recovery.

Effect of Ultrasound Therapy on Expression of Vascular Endothelial Growth Factor, Vascular Endothelial Growth Factor Receptors, CD31 and Functional Recovery After Facial Nerve Injury.

Functional recovery is provided by some neurotrophic factors released from the near vicinity of the injury site. Ultrasound treatment is known to increase neurotrophic factor expression. This study was aimed at determining the effect of ultrasound treatment on the expression of vascular endothelial growth factor (VEGF), its receptors and new vessel formation after facial nerve injury. Sixty-four Wistar rats were divided into four groups: control (group 1), sham (group 2), facial-facial coaptation (group 3), and facial-facial coaptation and ultrasound treatment (group 4). Animals in each group were evaluated on the 14th and 28th days. Immunohistochemical staining and electrophysiological and gene-level evaluations were performed for the expression of VEGF and its receptors. When the results were evaluated, it was determined that VEGF, VEGFR1 (VEGF receptor 1), VEGFR2 (VEGF receptor 2) and CD31 levels were significantly higher in groups 3 and 4 compared with the control and sham groups. The increase in these values was more prominent after 28 d of ultrasound treatment than all groups. Electrophysiological results revealed similar evident functional improvement in group 4 with decreased latency and increased amplitudes compared with group 3. Our findings suggest that ultrasound treatment might promote injured facial nerve regeneration by stimulating release of VEGF and its receptors and may result in functional improvement.

Infarct-preconditioning exosomes of umbilical cord mesenchymal stem cells promoted vascular remodeling and neurological recovery after stroke in rats

BackgroundStroke is the leading cause of disability worldwide, resulting in severe damage to the central nervous system and disrupting neurological functions. There is no effective therapy for promoting neurological recovery. Growing evidence suggests that the composition of exosomes from different microenvironments may benefit stroke. Therefore, it is reasonable to assume that exosomes secreted in response to infarction microenvironment could have further therapeutic effects.MethodsIn our study, cerebral infarct tissue extracts were used to pretreat umbilical cord mesenchymal stem cells (UCMSC). Infarct-preconditioned exosomes were injected into rats via tail vein after middle cerebral artery occlusion (MCAO). The effect of infarct-preconditioned exosomes on the neurological recovery of rats was examined using Tunel assay, 2,3,5-triphenyltetrazolium chloride (TTC) assay, magnetic resonance imaging (MRI) analyses, modified Neurological Severity Score (mNSS), Morris water maze (MWM), and vascular remodeling analysis. Mi-RNA sequencing and functional enrichment analysis were used to validate the signal pathway involved in the effect of infarct-preconditioned exosomes. Human umbilical vein endothelial cells (HUVECs) were co-cultured with the isolated exosomes. Cell Counting Kit-8 (CCK-8) assay, scratch healing, and Western blot analysis were used to detect the biological behavior of HUVECs.ResultsThe results showed that compared with normal exosomes, infarct-preconditioned exosomes further promoted vascular remodeling and recovery of neurological function after stroke. The function of upregulated miRNAs and their target genes which is beneficial to vascular smooth muscle cells verified the importance of vascular remodeling in improving stroke. Better resistance to oxygen–glucose deprivation/reoxygenation (OGD/R), reduced apoptosis, and enhanced migration were observed in infarct-preconditioned exosomes-treated umbilical vein endothelial cells.ConclusionsOur results demonstrated that infarct-preconditioned exosomes promoted neurological recovery after stroke by enhancing vascular endothelial remodeling, suggested that infarct-preconditioned exosomes could be a novel way to alleviate brain damage following a stroke.

A TRPC3/6 Channel Inhibitor Promotes Arteriogenesis after Hind-Limb Ischemia.

Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels.

Persistent vascular congestion in male spontaneously hypertensive rats contributes to delayed recovery of renal function following renal ischemia perfusion compared with females.

Acute kidney injury (AKI) due to ischemia is a serious and frequent clinical complication with mortality rates as high as 80%. Vascular congestion in the renal outer medulla occurs early after ischemia reperfusion (IR) injury, and congestion has been linked to worsened outcomes following IR. There is evidence implicating both male sex and preexisting hypertension as risk factors for poor outcomes following IR. The present study tested the hypothesis that male spontaneously hypertensive rats (SHR) have greater vascular congestion and impaired renal recovery following renal IR vs. female SHR and normotensive male Sprague-Dawley rats (SD). Thirteen-week-old male and female SHR and SD were subjected to sham surgery or 30 min of warm bilateral ischemia followed by reperfusion. Rats were euthanized 24 h or 7 days post-IR. IR increased renal injury in all groups vs. sham controls at 24 h. At 7 days post-IR, injury remained elevated only in male SHR. Histological examination of SD and SHR kidneys 24 h post-IR showed vascular congestion in males and females. Vascular congestion was sustained only in male SHR 7 days post-IR. To assess the role of vascular congestion on impaired recovery following IR, additional male and female SHR were pretreated with heparin (200 U/kg) prior to IR. Heparin pretreatment reduced IR-induced vascular congestion and improved renal function in male SHR 7 days post-IR. Interestingly, preventing increases in blood pressure (BP) in male SHR did not alter sustained vascular congestion. Our data demonstrate that IR-induced vascular congestion is a major driving factor for impaired renal recovery in male SHR.

Endothelial Mineralocorticoid Receptors Constrain Arteriogenesis and Perfusion Recovery Following Chronic Arterial Ligation in a Sex‐specific Manner

Vascular occlusive diseases are major contributors to cardiovascular morbidity and mortality. Prior work has implicated mineralocorticoid receptor (MR) signaling in perfusion recovery following arterial occlusion, however, cell‐specific MR signaling in the associated angiogenic and arteriogenic responses has not been elucidated. Further, accumulating evidence has revealed sex‐specific involvement of MR signaling in a number of cardiovascular disease states. Accordingly, we examined the hypothesis that MR signaling, specifically endothelial cell (EC) MR signaling, attenuates vascular compensation and perfusion recovery following femoral artery excision and that this effect would be more pronounced in females. Unilateral femoral artery ligation and excision was performed in male and female C57BL/6J mice treated with vehicle or the MR antagonist spironolactone (Spiro; 20 mg/kg/d, sc) as well as in male and female MR Intact and EC MR knockout (KO) mice. Serial assessment of hindlimb perfusion for 28 days following surgery revealed enhanced perfusion recovery following Spiro treatment in female, but not male, mice. Interestingly, Spiro increased gastrocnemius capillary density in both the ischemic and sham limb in males and females. Following perfusion fixation, collateral remodeling was assessed by micro‐computed tomography revealing enhanced collateral expansion following Spiro treatment in female, but not male, mice. Additional mechanistic experiments similarly revealed increased perfusion recovery and enhanced collateral expansion in female, not male, EC MR KO mice independent of capillary density. At 28 days post‐surgery, distribution analysis revealed larger overall arteriolar diameters only in the collateral zone of EC MR KO females. Further analysis revealed enhanced early (7 days post‐surgery) collateral expansion in male and female EC MR KO mice. In female, but not male, EC MR KO mice this was associated with increased CD68+ macrophages and CD206+ ‘M2’‐like macrophages in the collateral zone. Together, these data demonstrate that EC MR signaling constrains early arteriogenic collateralization following arterial excision in both sexes and constrains long‐term collateralization only in females involving limitation of pro‐arteriogenic CD206+ ‘M2’‐like macrophages in the collateral zone. To our knowledge, this is the first report of a role for EC MR signaling in arteriogenesis and highlights potential use of MR blockers in the treatment of vascular occlusive events.

Longitudinal dynamics of microvascular recovery after acquired cortical injury

Acquired brain injuries due to trauma damage the cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury, but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14 and 21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (> 200 μm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.

A Long-Term Clearing Cranial Window for Longitudinal Imaging of Cortical and Calvarial Ischemic Injury through the Intact Skull.

Skull is a reservoir for supplying immune cells that mediate brain immune surveillance. However, during intravital optical imaging of brain, conventional cranial windows requiring skull thinning or removal disrupt brain immunity integrity. Here, a novel long‐term clearing cranial window (LCCW) based on the intact skull, dedicated to chronic skull transparency maintenance, is proposed. It significantly improves optical imaging resolution and depth, by which the cortical and calvarial vascular injury and regeneration processes after ischemic injury are longitudinally monitored in awake mice. Results show that calvarial blood vessels recover earlier than the cortex. And the transcriptome analysis reveals that gene expression patterns and immune cells abundances exist substantial differences between brain and skull after ischemic injury, which may be one of the causes for the time lag between their vascular recovery. These findings bring great enlightenment to vascular regeneration and reconstruction. Moreover, LCCW provides a minimally invasive approach for imaging the brain and skull bone marrow.

Integration of vascular progenitors into functional blood vessels represents a distinct mechanism of vascular growth.

During embryogenesis, the initial vascular network forms by the process of vasculogenesis, or the specification of vascular progenitors de novo. In contrast, the majority of later-forming vessels arise by angiogenesis from the already established vasculature. Here, we show that new vascular progenitors in zebrafish embryos emerge from a distinct site along the yolk extension, or secondary vascular field (SVF), incorporate into the posterior cardinal vein, and contribute to subintestinal vasculature even after blood circulation has been initiated. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of the function of vascular progenitor marker etv2/etsrp prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. Similar late-forming etv2+ progenitors were also observed in mouse embryos, suggesting that SVF cells are evolutionarily conserved. Our results characterize a distinct mechanism by which new vascular progenitors incorporate into established vasculature.

Prolonged release of VEGF and Ang1 from intralesionally implanted hydrogel promotes perilesional vascularization and functional recovery after experimental ischemic stroke

Injectable hydrogels can generate and support pro-repair environments in injured tissue. Here we used a slow-releasing drug carrying in situ-forming hydrogel to promote post-stroke recovery in a rat model. Release kinetics were measured in vitro and in vivo with MRI, using gadolinium-labeled albumin (Galbumin), which demonstrated prolonged release over multiple weeks. Subsequently, this hydrogel was used for long-term delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) (Gel VEGF + Ang1, n = 14), in a photothrombotically induced cortical stroke lesion in rats. Control stroke animals were intralesionally injected with saline (Saline, n = 10), non-loaded gel (Gel, n = 10), or a single bolus of VEGF + Ang1 in saline (Saline VEGF + Ang1, n = 10). MRI was executed to guide hydrogel injection. Functional recovery was assessed with sensorimotor function tests, while tissue status and vascularization were monitored by serial in vivo MRI. Significant recovery from sensorimotor deficits from day 28 onwards was only measured in the Gel VEGF + Ang1 group. This was accompanied by significantly increased vascularization in the perilesional cortex. Histology confirmed (re)vascularization and neuronal sparing in perilesional areas. In conclusion, intralesional injection of in situ-forming hydrogel loaded with pro-angiogenic factors can support prolonged brain tissue regeneration and promote functional recovery in the chronic phase post-stroke.

Retinal Vascular Recovery Revealed by Retinal Imaging Following Neonatal Hypoxia Ischemia in Mice: Is There a Role for Tyrosine Kinase Receptor Modulation?

Retinal Vascular Recovery Revealed by Retinal Imaging Following Neonatal Hypoxia Ischemia in Mice: Is There a Role for Tyrosine Kinase Receptor Modulation?

Blood Pressure Variability Early After Liver Transplantation Predicts Long-Term Mortality.

Cardiovascular disease is a leading cause of mortality after liver transplantation (LT). Elevated blood pressure (BP) in LT recipients (LTRs) is associated with increased cardiovascular events (CVEs) and decreased survival. Increased visit-to-visit BP variability in the general population is associated with adverse outcomes. Whether BP variability is associated with adverse outcomes in LTRs is unknown. We analyzed data from adult LTRs within a single large transplant center in the United States between 2010 and 2016. Day-to-day BPvariability within the first 60 days after LT was measured usingvariability independent of the mean (VIM). To assess the association between early post-LT BP variability and future CVEs or mortality, we used Cox proportional hazard regression. Among 512 LTRs (34.4% women; 10.7% Black; mean age, 56.5 years), increased systolic BP (SBP) variability was associated with a decreased risk of mortality (adjusted hazard ratio [aHR], 0.97/1 unit VIM; 95% confidence interval [CI], 0.94-0.99). This was particularly true for men (aHR, 0.94; 95% CI, 0.91-0.98), patients with pre-LT atherosclerotic cardiovascular disease (aHR, 0.95; 95% CI, 0.92-0.98), and patients without pre-LT diabetes mellitus (aHR, 0.96; 95% CI, 0.93-1.00). There was no significant effect of BP variability on CVEs. Results were consistent when competing risk analysis was used with death as the competing risk. Increased diastolic BP variability was not associated with a significant effect on CVEs (hazard ratio [HR], 0.96; 95% CI, 0.90-1.02) nor mortality (HR, 1.00; 95% CI, 0.95-1.06). Increased SBP variability, independent of mean BP, is associated with decreased mortality in LTRs. We postulate that increased BP variability reflects a better vascular recovery in patients undergoing LT, but further research is needed as to the mechanism underlying our observation.

DENYING TOTAL HIP ARTHROPLASTY IN SMOKERS, PATIENTS WITH ALCOHOL ABUSE OR IN PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUSES. WHY?

Nowadays total hip arthroplasty (THA) is one of the most successful surgical procedures in the world and the number of procedures performed is growing every year. However, its success largely depends on the absence of postoperative complications. Among the risk factors affecting the occurrence of postoperative complications are smoking, alcohol abuse, drug abuse, human immunodeficiency viruses (HIV), obesity, anemia, diabetes mellitus, malnutrition, rheumatoid arthritis, cardiovascular diseases, renal failure and dialysis, depression and anxiety. In the presented manuscript, such factors as HIV, smoking and alcohol abuse were considered. In smokers, bone regeneration slows down due to impaired bone metabolism and a slowdown in vascular recovery. Alcohol abuse affects human immunity, inhibiting T-helper cells, and also causing blood coagulation disorders. Alcohol abuse increases the risk of hospital complications, surgery related complications and general medical complications. Smoking can increase the risk of septiccomplications (lower respiratory tract infection, sepsis, urinary tract infection), myocardial infarction, risk of aseptic loosening of implants. Mortality was also higher in smokers compared to nonsmokers. HIV increases bone fragility, debilitation, rate of cardiovascular diseases and decreases the number of CD4+ cells in the blood, which directly affects the risk of periprosthetic joint infections and revision. All three factors increase the patient's lengthof stay in the hospital after THA. Currently, recommendations have been developed for preventive measures that need to be taken to reduce the risk of postoperative complications by performing primary THA. According to the recommendations, quitting smoking and drinking alcohol 4 weeks before THA will significantly reduce the risk of postoperative complications. For HIV-positive patients, antiretroviral therapy and subsequent assessing the viral load arerequired prior to THA. Preoperative care in this category of patients, undergoing primary THA, can reduce the risk of complications.

MTORC2 Activation Mediated by Mesenchymal Stem Cell-Secreted Hepatocyte Growth Factors for the Recovery of Lipopolysaccharide-Induced Vascular Endothelial Barrier.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by pulmonary microvascular endothelial barrier dysfunction. Mesenchymal stem cell-secreted hepatocyte growth factor (HGF) has positive effects of lipopolysaccharide- (LPS-) induced pulmonary endothelial barrier. Studies have exhibited the mammalian TORC1 (mTORC1) signaling is of potent angiogenesis effects. The mTOR protein kinase has two distinct multiprotein complexes mTORC1 and mTORC2 that regulate different branches of the mTOR network. However, detailed mTORC2 mechanisms of HGF protective effects remain poorly defined. Therefore, the aim of this study was to determine whether mTORC2 mediated protective effects of MSC-secreted HGF against LPS-induced pulmonary microvascular endothelial barrier dysfunction activated like mTORC1 activation. We introduced MSC-PMVEC coculture transwell system and recombinant murine HGF on LPS-induced endothelial cell barrier dysfunction in vitro and then explored potential mechanisms by lentivirus vector-mediated HGF, mTORC1 (raptor), and mTORC2 (rictor) gene knockdown modification. Endothelial paracellular and transcellular permeability, adherent junction protein (VE-Cadherin), cell proliferation, apoptosis, and mTOR-associated proteins were tested. These revealed that HGF could promote quick reestablishment of adherent junction VE-cadherin and decrease endothelial paracellular and transcellular permeability during LSP-induced endothelial dysfunction with the involvement of mTORC2 (rictor) and mTORC1 (raptor) pathways. Raptor and rictor knockdown in LPS-induced PMEVECs with stimulation of HGF increased apoptosis ratio, activated Cleaved-Caspase-3 expression, and downregulated cell proliferation. Moreover, mTORC2/Akt but not mTORC2/PKC had significance on HGF endothelial protective effects. Taken together, these highlight activation mTORC2 pathway could also contribute to vascular endothelial barrier recovery by MSC-secreted HGF in LPS stimulation.

MicroRNA-126 mimic administration accelerates vascular perfusion recovery and angiogenesis in a hind limb ischemia model

Abstract Background Peripheral arterial disease caused mainly by atherosclerosis portent significant morbidity, adverse prognosis and mortality, with localized treatment approaches aiming at symptom alleviation and improvement of circulation. Recently, scientific interest has been shifted towards epigenomics, with microRNAs appearing as a future therapeutic target in ischemic cardiovascular diseases due to their potential in regulating angiogenesis. Purpose We investigated the pro-angiogenic effect of miRNA-126 mimic in an in vivo model of hind limb ischemia. Methods Ten-week-old male C57Bl/6 mice (n=20) were subjected to left femoral artery ligation and were treated with microRNA-126 mimic at a dose of 5mg/kg (Group A, n=10) or 0.2ml normal saline (Group B, n=10) on days 1, 3 and 7. Laser Doppler imaging was performed to verify successful ligation on day 0 and to evaluate differences in the ischemic-to-normal (I/N) hind limb perfusion ratio on day 7 and 28. Muscle tissue expression of microRNA-126 and vascular endothelial growth factor (VEGF) was determined via PCR. Results Following microRNA-126 mimic administration in Group A subjects, we noted a qualitative and quantitative stepwise increase in I/N hind limb perfusion ratio [Day 0: 0.354 (0.276, 0.455) vs. Day 8: 0.775 (0.700, 0.844) vs. Day 28: 0.681 (0.660, 0.896), p=0.001] (Figure 1, Panels A and B). In Group B a stepwise increase of lesser magnitude was observed in I/N hind limb perfusion ratio [Day 0: 0.267 (0.164, 0.383) vs. Day 8: 0.400 (0.338, 0.418) vs. Day 28: 0.539 (0.483, 0.603), p=0.074]. Importantly, over time changes of I/N hind limb perfusion ratio were significantly higher in group A compared to group B (p for interaction=0.005) (Figure 1, Panel B). Muscle tissue expression of microRNA-126 in the ischemic hind limb of Group A was 350-fold lower compared to the ischemic hind limb of Group B (p<0.001) (Figure 1, Panel C). A higher expression (14.2-fold) of VEGF in the ischemic hind limb of microRNA-126-treated mice compared to that of control group was detected (p<0.001) (Figure 1, Panel C). A statistically significant negative correlation was noted between microRNA-126 and VEGF tissue expression levels in the ischemic limbs of both Group A and B subjects whereas no correlation between microRNA-126 and VEGF was observed in the non-ischemic hind limbs of the entire study population (Figure 1, Panel D). Conclusion MicroRNA-126 mimic delivery in the ischemic hind limb of mice can accelerate vascular perfusion recovery via angiogenesis, which is mediated by VEGF expression. Funding Acknowledgement Type of funding sources: None. Figure 1

Exosomal OTULIN from M2 macrophages promotes the recovery of spinal cord injuries via stimulating Wnt/β-catenin pathway-mediated vascular regeneration

Vascularization following spinal cord injury (SCI) provides trophic support for rebuilding up and maintaining the homeostasis of neuronal networks, and the promotion of angiogenesis is beneficial for functional recovery after SCI. M2 macrophages have been reported to exhibit powerful pro-angiogenic functions during tissue repair. Exosomes are important paracrine mediators of their parent cells and play critical roles in tissue regeneration. However, the role of M2 macrophage-derived exosomes (M2-Exos) in SCI is still largely unknown. In the present study, we determined that M2-Exos could augment the angiogenic activities of spinal cord microvascular endothelial cells (SCMECs) in vitro. Hydrogel-mediated sustained release of M2-Exos significantly promoted vascular regeneration and functional recovery in mice after SCI. Furthermore, proteomics analysis showed that ubiquitin thioesterase otulin (OTULIN) protein was highly enriched in M2-Exos. Functional assays demonstrated that OTULIN protein was required for the M2-Exos-induced pro-angiogenic effects in SCMECs, as well as positive effects on vascular regeneration, cell proliferation, and functional recovery in the mouse model of SCI. Mechanically, OTULIN from M2-Exos could activate the Wnt/β-catenin signaling by increasing the protein level of β-catenin via inhibiting its ubiquitination and trigger the expression of angiogenesis-related genes that are reported to be the downstream targets of Wnt/β-catenin signaling. Inhibition of the Wnt/β-catenin signaling by ICG001 markedly attenuated the pro-angiogenic activities of M2-Exos in vitro/vivo. Our findings indicate that M2-Exos positively modulate vascular regeneration and neurological functional recovery after SCI by activating Wnt/β-catenin signaling through the transfer of OTULIN protein. Statement of significanceM2 macrophages have been identified to promote vascular regeneration, cell proliferation and tissue growth after spinal cord injury (SCI), which is beneficial to the functional recovery. Exosomes are essential paracrine mediators involved in cell-to-cell communication and play important roles in tissue regeneration. In the present study, we revealed that M2 macrophages-derived exosomes (M2-Exos) could promote functional recovery post SCI by targeting angiogenesis. We demonstrated for the first time that OTULIN protein from M2-Exos mediated the angiogenic effects through activating Wnt/β-catenin signaling and triggering the expression of angiogenic-related genes in spinal cord microvascular endothelial cells (SCMECs). The hydrogel-M2-Exos sustained released system provides potential therapeutic clues of local cell-free interventions for the treatment of SCI.

Expanding the limits of TAVI: First in man report of valve implantation in a 25-year-old patient with mucolipidosis

Transcatheter aortic valve implantation (TAVI) is commonly used in older patients with aortic stenosis considered at high or intermediate risk for surgical replacement. To our knowledge, TAVI has not been used in young patients or in patients with paediatric dimensions. We report a case of a 25-year-old patient followed since childhood for mucolipidosis type III with thoraco-lombar cyphosis, chronic kidney disease and evolving aortic regurgitation (AR). Two acute heart failure episodes occurred during the last year requiring intravenous loop diuretics, on top of chronic dyspnea (NYHA III–IV). Patient height was 114 cm and weight 27 kg. Echocardiography showed severe aortic regurgitation with dilated LV (Z-value LVED + 4.5) and LV dysfunction (LVEF = 35%). The annulus was measured at 21 mm. TAVI was considered as surgery was considered at too high-risk. CT scan showed appropriate aortic valve anatomy but also a tortuous abdominal aorta ( Fig. 1 ). Femoral access was obtained under loco-regional anaesthesia, and heparin was administered. A Corevalve EVOLUT™ R 29 mm valve (Medtronic) was deployed, using rapid pacing ( Fig. 2 ). Post-implantation echocardiography showed no significant leak, patent coronary arteries and a satisfactory transvalvular gradient at 7 mmHg. The procedure was complicated by a left bundle branch block without indication for a pacemaker. Femoral access was complicated by asymptomatic thrombosis of the external illiac artery with good vascular recovery. At last follow-up, patient was improved (NYHA II). Echocardiography showed good position of aortic prosthesis, transvalvular gradient at 4 mmHg without aortic regurgitation or paravalvular leak. Our case report confirms that TAVI may be considered for young adults or patients with paediatric dimensions, in whom surgery is at too high-risk, due to important comorbidities. Vascular access remains an important issue when dealing with small patients.

The Effect of MicroRNA-126 Mimic Administration on Vascular Perfusion Recovery in an Animal Model of Hind Limb Ischemia.

Background: MicroRNAs have been linked to angiogenesis and could prove to be valuable future therapeutic targets in ischemic cardiovascular diseases.Methods: Ten-week-old male C57Bl/6 mice were subjected to left femoral artery ligation and were treated with microRNA-126 mimic at a dose of 5 mg/kg (Group A, n = 10) or 5 mg/kg microRNA mimic negative control (Group B, n = 10) on days 1, 3, and 7. Laser Doppler imaging was performed to verify successful ligation on day 0 and to evaluate differences in the ischemic-to-normal (I/N) hind limb perfusion ratio on day 28. Muscle tissue expression of microRNA-126 and vascular endothelial growth factor (VEGF) was determined via PCR.Results: Following microRNA-126 mimic administration in Group A subjects, we noted a stepwise increase in I/N hind limb perfusion ratio (Day 0: 0.364 ± 0.032 vs. Day 8: 0.788 ± 0.049 vs. Day 28: 0.750 ± 0.039, p = 0.001). In Group B a stepwise increase in I/N hind limb perfusion ratio was observed (Day 0: 0.272 ± 0.057 vs. Day 8: 0.382 ± 0.020 vs. Day 28: 0.542 ± 0.028, p = 0.074). Muscle tissue expression of microRNA-126 in the ischemic hind limb of Group A was 350-fold lower compared to the ischemic hind limb of Group B (p < 0.001). A higher expression (14.2-fold) of VEGF in the ischemic hind limb of microRNA-126-treated mice compared to that of control group was detected (p < 0.001). A statistically significant negative correlation was noted between microRNA-126 and VEGF tissue expression levels in the ischemic limbs of the entire study population.Conclusion: MicroRNA-126 delivery in the ischemic hind limb of mice improved vascular perfusion with VEGF upregulation.