Vascular Expression Research Articles

Inflammatory Forms of Cardiomyocyte Cell Death in the Rat Model of Isoprenaline-Induced Takotsubo Syndrome.

Takotsubo syndrome (TTS) is associated with inflammatory response, therefore the aim of the study was to evaluate the presence and dynamics of inflammatory-associated forms of cell death, necroptosis, and pyroptosis in the female rat model of isoprenaline (ISO)-induced TTS. TTS was induced in female Sprague Dawley rats (n = 36) by ISO 150 mg/kg intraperitoneally. Animals were divided into four groups: TTSO (TTS+ovariectomy; n = 10), TTSP (TTS+sham operation; n = 10), CO (0.9% NaCl+ovariectomy; n = 8), CP (0.9% NaCl+sham operation; n = 8). Histopathological analysis, evaluation of plasma concentration, and myocardial expression of pyroptosis- and necroptosis-associated proteins were performed. TTSO and TTSP groups had higher plasma concentrations of interleukin-1β in comparison with the controls. Low myocardial protein expression of mixed lineage kinase domain-like pseudokinase (MLKL), caspase-1 (Casp-1), and calcium/calmodulin-dependent kinase type II isoform delta (CAMKIIδ) was visible 6 and/or 12 h post-ISO. Twenty-four hours post-ISO, high myocardial and vascular protein expression of CAMKIIδ was visible in TTSO but not TTSP rats, while high myocardial expression of MLKL and Casp-1 was visible both in TTSO and TTSP rats. The course of TTS is associated with activation of inflammatory-associated programmed cell death, necroptosis, and pyroptosis, therefore inflammation may be a primary response occurring simultaneously with cardiomyocyte death in TTS.

Vascular Expression of Prostate-specific Membrane Antigen (PSMA) in MiTF Family Translocation Renal Cell Carcinoma and Related Neoplasms.

Multiple studies have demonstrated prostate-specific membrane antigen (PSMA) expression in the neo-vasculature of non-prostate tumors including clear cell renal cell carcinoma (ccRCC). However, PSMA expression in rare renal tumors including MiTF family translocation renal cell carcinoma has not been previously characterized. We examined PSMA expression by immunohistochemistry in a series of MiTF family translocation renal cell carcinomas as well as in several genetically related tumors including alveolar soft part sarcoma and PEComas with TFE3 rearrangements. PSMA expression was also studied in several cases of ccRCC and papillary RCC. Overall, PSMA immunohistochemistry was performed in 61 samples from 58 patients. Vascular PSMA expression was seen with the highest frequency in ccRCC [88% (14/16)] (38% focal, 50% diffuse). Translocation RCC (tRCC) demonstrated the second highest frequency of PSMA expression [71% (22/28)] (57% focal, 14% diffuse), followed by alveolar soft part sarcoma [50% (4/8)] (38% focal, 12% diffuse). No PSMA expression was seen in PEComas with TFE3 rearrangement (0/3) or papillary RCC (0/6). PSMA expression was only present in tumor-associated neo-vasculature. A patient with oligometastatic tRCC underwent 68 Ga-PSMA-11 PET imaging which detected multiple putative metastatic lesions not detected on conventional computed tomography imaging performed 2 weeks prior, supporting the potential utility of PSMA imaging in tRCC. These findings have potential implications for the utility of PSMA guided diagnostic and therapeutic agents in both common and uncommon renal cell carcinoma subtypes as well as genetically related mesenchymal neoplasms.

Carbamoylated Erythropoietin-Induced Cerebral Blood Perfusion and Vascular Gene Regulation.

Cerebral hypoperfusion is associated with enhanced cognitive decline and increased risk of neuropsychiatric disorders. Erythropoietin (EPO) is a neurotrophic factor known to improve cognitive function in preclinical and clinical studies of neurodegenerative and psychiatric disorders. However, the clinical application of EPO is limited due to its erythropoietic activity that can adversely elevate hematocrit in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered non-erythropoietic derivative of EPO, does not alter hematocrit and maintains neurotrophic and behavioral effects comparable to EPO. Our study aimed to investigate the role of CEPO in cerebral hemodynamics. Magnetic resonance imaging (MRI) analysis indicated increased blood perfusion in the hippocampal and striatal region without altering tight junction integrity. In vitro and in vivo analyses indicated that hippocampal neurotransmission was unaltered and increased cerebral perfusion was likely due to EDRF, CGRP, and NOS-mediated vasodilation. In vitro analysis using human umbilical vein endothelial cells (HUVEC) and hippocampal vascular gene expression analysis showed CEPO to be a non-angiogenic agent which regulates the MEOX2 gene expression. The results from our study demonstrate a novel role of CEPO in modulating cerebral vasodilation and blood perfusion.

The secreted neuronal signal Spock1 promotes blood-brain barrier development

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 initiates BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.

Itaconic acid induces angiogenesis and suppresses apoptosis via Nrf2/autophagy to prolong the survival of multi-territory perforator flaps

BackgroundPerforator flaps are widely used in hand microsurgery to reconstruct and repair soft tissue injuries. However, ischemia and subsequent ischemia-reperfusion injury may cause distal necrosis of the flap. Itaconic acid (IA) is a modulator of macrophage function, which exerts anti-inflammatory effects in macrophage activation. MethodsThe necrotic area of the flap was detected by measuring the flap temperature with an infrared thermometer. Flap cell apoptosis was detected by TUNEL staining and Western blot analysis of the apoptosis-associated proteins Bcl-2 and Bax. HE staining was used to detect angiogenesis of the skin flaps. CD31 was detected to identify positive vascular expression, and the survival of choke vessels in different areas of the skin flap was assessed by arteriography. In addition, Western blot was performed to quantify the expressions of VEGF, Nrf2, LC3II, SQSTM1, and CTSD. ResultsItaconic acid raises VEGF protein levels in skin flaps and the number of CD31-positive vessels. The skin flaps in the IA treatment group exhibited higher neovascularization and less necrosis than those in the control group. The results of TUNEL staining and Western blot revealed that Itaconic acid attenuated apoptosis in the ischemic area of flap. The combination of itaconic acid and Nrf2 inhibitor ML385 reversed this beneficial effect. The results revealed that itaconic acid attenuated apoptosis, enhanced angiogenesis, and enhanced autophagy. ConclusionIn summary, our findings indicate that itaconic acid might be an effective treatment to reduce flap necrosis. Additionally, this study identified a novel mechanism for the effects of itaconic acid on flap survival.

First waikavirus infectious clones and vascular expression of green fluorescent protein from maize chlorotic dwarf virus

Plant viruses classified in the genus Waikavirus, family Secoviridae, are positive sense single-stranded RNA viruses that include important pathogens of maize (maize chlorotic dwarf virus; MCDV) and rice (rice tungro spherical virus; RTSV). Many aspects of the molecular biology of waikaviruses remain unexplored because of experimental challenges and lack of infectious clones for low titer, phloem-limited, and obligately vector-transmitted waikaviruses. Here we report the first development of waikavirus infectious clones for two MCDV strains, MCDV-S and MCDV-M1, and insect-free launching of infections from these clones in maize by vascular puncture inoculation. We further developed a green fluorescent protein (GFP)-tagged MCDV clone by replacing the viral p27-encoding sequence with GFP-encoding sequence. GFP-tagged virus moved systemically in plants and caused symptomatic infection similar to wild type virus, with vascular expression of GFP. Development of waikavirus infectious clones is a major advance for this group of agriculturally significant viruses.

Nano hydrogel-based oxygen-releasing stem cell transplantation system for treating diabetic foot

The employment of stem cells and hydrogel is widespread in contemporary clinical approaches to treating diabetic foot ulcers. However, the hypoxic conditions in the surrounding lesion tissue lead to a low stem cell survival rate following transplantation. This research introduces a novel hydrogel with superior oxygen permeability and biocompatibility, serving as a vehicle for developing a stem cell transplantation system incorporating oxygen-releasing microspheres and cardiosphere-derived stem cells (CDCs). By optimizing the peroxidase fixation quantity on the microsphere surface and the oxygen-releasing microsphere content within the transplantation system, intracellular oxygen levels were assessed using electron paramagnetic resonance (EPR) under simulated low-oxygen conditions in vitro. The expression of vascularization and repair-related indexes were evaluated via RT-PCR and ELISA. The microspheres were found to continuously release oxygen for three weeks within the transplantation system, promoting growth factor expression to maintain intracellular oxygen levels and support the survival and proliferation of CDCs. Moreover, the effect of this stem cell transplantation system on wound healing in a diabetic foot mice model was examined through an in vivo animal experiment. The oxygen-releasing microspheres within the transplantation system preserved the intracellular oxygen levels of CDCs in the hypoxic environment of injured tissues. By inhibiting the expression of inflammatory factors and stimulating the upregulation of pertinent growth factors, it improved the vascularization of ulcer tissue on the mice’s back and expedited the healing of the wound site. Overall, the stem cell transplantation system in this study, based on hydrogels containing CDCs and oxygen-releasing microspheres, offers a promising strategy for the clinical implementation of localized stem cell delivery to improve diabetic foot wound healing.

458-P: Proinflammatory Macrophage Phenotype Modulates Vascular Smooth Muscle Cell Differentiation via the Histone Demethylase JMJD3

Patients with diabetes are at increased risk of a multitude of vascular diseases, including atherosclerosis, which is driven by macrophage-mediated inflammation. While it is known that atherosclerosis is characterized by a proliferative, synthetic smooth muscle phenotype that underlies disease, the exact role of other cell types, namely myeloid cells and their interaction with smooth muscle cells is less understood. Furthermore, how diabetes modifies atherosclerotic severity is another important question. To address this, we performed an epigenetic superarray to identify key chromatin modifying enzymes in vascular smooth muscle cells (vSMC) from normal diet (ND) and diabetic-induced obese (DIO) aortas. We identified the histone demethylase, JMJD3, which was significantly decreased in DIO compared to ND vSMC (p<0.05). Furthermore, in our single cell RNA-seq data set from human atherosclerotic lesions, JMJD3 was expressed in smooth muscle cells. It is known that macrophages in atherosclerotic lesions exhibit a pro-inflammatory phenotype. To model this pro-inflammatory process, we stimulated bone marrow derived macrophages with LPS and found that supernatant from LPS-stimulated BMDMs significantly inhibited smooth muscle specific gene expression (SMA, SM22, CNN1, SM-MHC) as well as expression of JMJD3 compared to control media (p<0.05). Additionally, siRNA-mediated knockdown of JMJD3 in vSMC reduced SMC-specific gene expression, and treatment of mouse aortic vSMC with the JMJD3 selective inhibitor, GSK4J, decreased smooth muscle gene expression (p<0.05). Our data shows that pro-inflammatory macrophages drive reduced vascular smooth muscle gene expression and that JMJD3 in SMC is a regulator of this process. These results implicate JMJD3 in macrophage-smooth muscle cell crosstalk in the pro-inflammatory state and identify it as a potential therapeutic target in the management of atherosclerosis. Disclosure K.D.Mangum: None. A.Joshi: None. P.Mazumder: None. S.Wolf-fortune: None. E.Barrett: None. B.Moore: None. F.M.Davis: None. K.Gallagher: None. Funding National Institutes of Health (1F32DK131799-01); American College of Surgeons; Vascular & Endovascular Surgery Society

CTLA-4 and PD-1 blocking agents affects longitudinal and radial strain in preclinical models, increases systemic SDF-1, cardiac fibronectin, S-100 calgranulin, galectine-3 and NLRP-3/MyD-88 pathways

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca Corrente, Ministero della Salute. Background Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro-inflammatory and pro-fibrotic effects of short-term ICIs therapy in preclinical models were analyzed. Methods Firstly, in a human in vitro model, human cardiomyocytes co-cultured with hPBMC were exposed to ICIs (with CTLA-4 or PD-1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA-4 or PD-1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF-kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100- Calgranulin, Fibronectin and Galectine-3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF-1, IL-1β and IL-6 were analyzed before, during and after ICIs therapy. Results Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF-kB expression shows that short-term anti-CTLA-4 or anti-PD-1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin-3, pro-collagen 1-α and MMP-9 were increased after treatment with all ICIs. Both anti-CTLA-4 or anti-PD-1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL-1β, TNF-α and IL-6. Systemic levels of SDF-1, IL-1β and IL-6 were increased during and after treatment with ICIs. Conclusions Short therapy with PD-1 and CTLA-4 blocking agents increases vascular expression of NF-kB, systemic SDF-1, IL-1β, IL-6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro-inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy. The overall picture of the study suggests new putative biomarkers of ICIs-mediated systemic and myocardial damages potentially useful in clinical cardioncology.

A role for P-selectin and complement in the pathological sequelae of germinal matrix hemorrhage

BackgroundGerminal matrix hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH.MethodsWe prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv’s) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal C57BL/6 J mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle.ResultsCompared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. In addition, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet–leukocyte aggregation, which express P-selectin and PSGL-1, respectively.ConclusionsGMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

Facile synthesis of zinc acetate/niacin MOFs for use in wound healing

Niacin (NA) and zinc (Zn) were used to fabricate metal organic frameworks (Zn-NA MOFs), based on coordination chemistry via a simple, rapid technique conducted at room temperature. The identity of the prepared MOFs was confirmed by Fourier-transform infrared, x-ray diffraction, scanning electron microscopy, and transmission electron microscopy, which showed cubic shaped, crystalline, microporous MOFs with an average size of 150 nm. Release of the active ingredients from the MOFs was proved to be pH dependent in a slightly alkaline medium (pH 8.5) with a sustained release rate of its two ingredients, NA and Zn, which have wound healing activity. Zn-NA MOFs proved to be biocompatible in the tested concentrations range (5–100 mg ml−1), with no cytotoxic effect on WI-38 cell line. Zn-NA MOFs at 10 and 50 mg ml−1 concentrations and their components, NA and Zn, exerted antibacterial effects against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Wound healing effect of the Zn-NA MOFs (50 mg ml−1) was evaluated on full excisional rat wounds. Significant reduction of the wound area was observed after 9 d of treatment using the Zn-NA MOFs compared to the other treatment groups. Additionally, wounds were fully healed after 10 d of treatment with the Zn-NA MOFs with histological and immunohistochemical evidence of re-epithelization, collagen formation, and angiogenesis. Similar histological evidence was also observed in wounds treated with niacin only; however, with no significant wound closure rates. Nevertheless, the formation of new blood vessels, as confirmed by the vascular endothelial growth factor protein expression, was highest in the niacin group. Zn-NA MOFs synthesized using a facile, low-cost method are potentially capable of healing wounds rapidly and effectively.

Extrinsic Vascular Pathway Preservation Improves Survival in a Rat Three-Territory Flap Model Based on the Deep Circumflex Iliac Artery.

Extended flaps are commonly applied for large defects. However, a postoperative flap necrosis incidence of 11% to 44% remains a major complication. Previous clinical studies have shown that maintaining the extrinsic vascular pathway (EVP) can increase the survival area of extended flaps. The authors hypothesized that preserving the EVP would improve flap survival by reducing blood resistance within the vascular territory. Twenty-four adult male Sprague-Dawley rats were used. Tissue samples were obtained from eight untreated rats as a baseline control. Three-territory flaps were elevated in the remaining 16 rats. The EVP was preserved or ligated. Flap perfusion was assessed immediately using indocyanine green angiography. Rats were euthanized on day 7. The flap survival area was measured using Adobe Photoshop. Hematoxylin and eosin staining, CD31 immunostaining, and Western blot analysis of vascular endothelial growth factor protein expression were used to quantitatively assess vasodilation and angiogenesis in choke zones. Indocyanine green angiography revealed that blood could flow through the preserved EVP and perfuse the third vascular territory of the flap. EVP preservation significantly increased flap survival area (86.3%, 19.3% difference; P < 0.001), promoted vasodilation (5.0/choke zone, 3.0/choke zone difference; P = 0.013) and angiogenesis (29.3/mm 2 , 14.3/mm 2 difference; P = 0.002), and increased vascular endothelial growth factor expression (0.6, 0.2 difference; P = 0.067) in the second choke zone. EVP preservation improves flap survival in this rat three-territory flap model. Further investigation in large-animal models is required for clinical translation. Although further validation in large animal models and prospective clinical trials are necessary to verify the efficacy of the authors' hypothesis, their findings suggest that the EVP preservation procedure could provide an alternative for surgeons to create an extended flap in defect reconstruction.

P128 CTLA–4 AND PD–1 BLOCKING AGENTS AFFECTS LONGITUDINAL AND RADIAL STRAIN IN PRECLINICAL MODELS, INCREASES SYSTEMIC CXCL12, CARDIAC FIBRONECTIN EDA, S–100 CALGRANULIN, GALECTINE–3 AND NLRP–3/MYD–88/CHEMOKINE PATHWAYS

Abstract Background Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro–inflammatory and pro–fibrotic effects of short–term ICIs therapy in preclinical models were analyzed. Methods Firstly, in a human in vitro model, human cardiomyocytes co–cultured with hPBMC were exposed to ICIs (with CTLA–4 or PD–1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA–4 or PD–1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF–kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100– Calgranulin, Fibronectin and Galectine–3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF–1, IL–1β and IL–6 were analyzed before, during and after ICIs therapy. Results Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF–kB expression shows that short–term anti–CTLA–4 or anti–PD–1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin–3, pro–collagen 1–α and MMP–9 were increased after treatment with all ICIs. Both anti–CTLA–4 or anti–PD–1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL–1β, TNF–α and IL–6. Systemic levels of SDF–1, IL–1β and IL–6 were increased during and after treatment with ICIs. Conclusions Short therapy with PD–1 and CTLA–4 blocking agents increases vascular expression of NF–kB, systemic SDF–1, IL–1β, IL–6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro–inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy.

Graphdiyne-loaded polycaprolactone nanofiber scaffold for peripheral nerve regeneration

Graphdiyne (GDY) is a kind of nanomaterial from the graphene carbon family with excellent physical and chemical properties. Despite some applications in medical engineering, GDY has not been used as an electroactive scaffold for tissue regeneration because of its unclear in vitro and in vivo biosafety profiles. Here, a conductive GDY nanomaterial-loaded polycaprolactone (PCL) scaffold was prepared by electrospinning technique. For the first time, the biocompatibility of GDY-based scaffold was assessed at the cellular and animal levels in a peripheral nerve injury (PNI) model. The findings indicated that the conductive three-dimensional (3D) GDY/PCL nerve guide conduits (NGCs) could significantly improve the proliferation, adhesion and glial expression of Schwann cells (SCs). The conduits were implanted into a rat 10-mm sciatic nerve defect model for 3 months in vivo. The toxicity of scaffolds to the organs was negligible, while the GDY/PCL NGCs significantly promoted myelination and axonal growth by upregulating the expression levels of SC marker (S100 β protein), Myelin basic protein (MBP), and axon regeneration marker (β3-tubulin protein (Tuj1) and neurofilament protein 200 (NF200)). In addition, upregulation of vascular factor expression in GDY/PCL NGC group suggested the potential role in angiogenesis to improve nerve repair by GDY nanomaterials. Our findings provide new perspectives on biocompatibility and effectiveness of GDY nanomaterial scaffold in peripheral nerve regeneration for preclinical application.

Anti-Fibrotic Efficacy of Apigenin in a Mice Model of Carbon Tetrachloride-Induced Hepatic Fibrosis by Modulation of Oxidative Stress, Inflammation, and Fibrogenesis: A Preclinical Study.

Hepatic fibrosis is a major health problem all over the world, and there is no effective treatment to cure it. Hence, the current study sought to assess the anti-fibrotic efficacy of apigenin against CCl4-induced hepatic fibrosis in mice. Forty-eight mice were put into six groups. G1: Normal Control, G2: CCl4 Control, G3: Silymarin (100 mg/kg), G4 and G5: Apigenin (2 &20 mg/Kg), G6: Apigenin alone (20 mg/Kg). Groups 2, 3, 4, and 5 were given CCl4 (0.5 mL/kg. i.p.) twice/week for six weeks. The level of AST, ALT, TC, TG, and TB in serum and IL-1β, IL-6, and TNF-α in tissue homogenates were assessed. Histological studies by H&E staining and Immunostaining of liver tissues were also performed. The CCl4-challenged group showed increased serum AST (4-fold), ALT (6-fold), and TB (5-fold). Both silymarin and apigenin treatments significantly improved these hepatic biomarkers. The CCl4-challenged group showed reduced levels of CAT (89%), GSH (53%), and increased MDA (3-fold). Both silymarin and apigenin treatments significantly altered these oxidative markers in tissue homogenates. The CCl4-treated group showed a two-fold increase in IL-1β, IL-6, and TNF-α levels. Silymarin and apigenin treatment considerably decreased the IL-1β, IL-6, and TNF-α levels. Apigenin treatment inhibited angiogenic activity, as evidenced by a decrease in VEGF (vascular endothelial growth factor) expression in liver tissues, and a decline in vascular endothelial cell antigen expression (CD34). Finally, these data collectively imply that apigenin may have antifibrotic properties, which may be explained by its anti-inflammatory, antioxidant, and antiangiogenic activities.

Endothelial beta-globin is differentially expressed across mammalian species

Globin proteins interact with endothelial nitric oxide synthase at the myoendothelial junctions of resistance arteries to regulate nitric oxide signaling between endothelium and smooth muscle. Human resistance arteries express both alpha and beta globin to form heterotetrameric hemoglobin (α2β2). To better understand the role of hemoglobin in human resistance arteries, we need to identify an animal model in which both alpha and beta globin are expressed in the resistance vasculature. To identify potential candidates, we used microscopy to survey a range of mammalian laboratory animals for hemoglobin in the artery wall. We isolated arteries (100-200 μm in diameter) from mesenteric tissue collected from human patients undergoing clinically-indicated surgery and from the following laboratory animals: mouse ( Mus musculus, C57BL/6 strain), rat ( Rattus norvegicus, CD strain), African thicket rat ( Grammomys surdaster), guinea pig ( Cavia porcellus), New Zealand white rabbit ( Oryctolagus cuniculus), Beagle dog ( Canis lupus familiaris), and cynomolgus macaque ( Macaca fascicularis) after they underwent euthanasia on approved animal study protocols. We leveraged the autofluorescence of heterotetrameric hemoglobin under multiphoton excitation to visualize hemoglobin in the wall of intact, unstained, paraformaldehyde-fixed artery segments. Autofluorescent puncta representing tetrameric hemoglobin were observed in the wall of resistance arteries from the human patients, non-human primate, and canines, but not from rabbit nor any of the rodent species. The ability of beta globin to restrict endothelial NO signaling was confirmed in canine arteries ex vivo by testing a beta globin mimetic peptide that inhibited vasoconstriction to phenylephrine. In conclusion, primates and canines appear to have tetrameric hemoglobin in the resistance artery wall, whereas rodents and rabbits do not. This study highlights differences in vascular globin expression between humans and rodents and extends the recent discovery of hemoglobin in resistance arteries of humans to two additional mammalian species that may serve as models for studying the unique activities of tetrameric hemoglobin in the resistance vasculature. Funding: Intramural Research program, NIAID, NIH This is the full abstract presented at the American Physiology Summit 2023 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.

Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia

Objective: Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways. Methods: Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium. Results: Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p &lt;0.001) and matrix metalloprotease-2 (MMP-2, p &lt;0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p &gt;0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged. Conclusions: In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.

Therapeutic effects of a standardized-flavonoid Diospyros kaki L.f. leaf extract on transient focal cerebral ischemia-induced brain injury in mice.

This study aimed to investigate the neuroprotective and therapeutic effects of Diospyros kaki L.f. leaves (DK) on transient focal cerebral ischemic injury and underlying mechanisms using a middle cerebral artery occlusion (MCAO) model of mice. The animals received the MCAO operation on day 0. The daily administrations of DK (50 and 100mg/kg, p.o) and edaravone (6mg/kg, i.v), a reference drug with radical scavenging activity, were started 7days before (pre-treatment) or immediately after the MCAO operation (post-treatment) and continued during the experimental period. Histochemical, biochemical, and neurological changes and cognitive performance were evaluated. MCAO caused cerebral infarction and neuronal cell loss in the cortex, striatum, and hippocampus in a manner accompanied by spatial cognitive deficits. These neurological and cognitive impairments caused by MCAO were significantly attenuated by pre- and post-ischemic treatments with DK and edaravone, suggesting that DK, like edaravone, has therapeutic potential for cerebral ischemia-induced brain damage. DK and edaravone suppressed MCAO-induced changes in biomarkers for apoptosis (TUNEL-positive cell number and cleaved caspase-3 protein expression) and oxidative stress (glutathione and malondialdehyde contents) in the brain. Interestingly, DK, but not edaravone, mitigated an increase in blood-brain permeability and down-regulation of vascular endothelial growth factor protein expression caused by MCAO. Although the exact chemical constituents implicated in the effects of DK remain to be clarified, the present results indicate that DK exerts neuroprotective and therapeutic activity against transient focal cerebral ischemia-induced injury probably by suppressing oxidative stress, apoptotic process, and mechanisms impairing blood-brain barrier integrity in the brain.

A ketogenic diet improves vascular hyperpermeability in type 2 diabetic mice by downregulating vascular pescadillo1 expression.

The role of pescadillo1 (PES1) in regulating vascular permeability has been unknown. This study probes the role of PES1 and its mediated molecular mechanism in modulating vascular hyperpermeability in diabetic mice. Male C57BL/6J and db/db mice were fed a standard diet and a ketogenic diet (KD). Meanwhile, mouse vascular endothelial cells (MVECs) were treated with β-hydroxybutyric acid (β-HB), Pes1 siRNA or a Pes1 overexpression plasmid. Additionally, knockout (KO) of Pes1 in mice was applied. After 12 weeks of feedings, enhanced vascular PES1 expression in diabetic mice was inhibited by the KD. The suppression of PES1 was also observed in β-HB-treated MVECs. In mice with Pes1 KO, the levels of vascular VEGF and PES1 were attenuated, while the levels of vascular VE-cadherin, Ang-1 and Occludin were upregulated. Similar outcomes also occurred after the knockdown of Pes1 in cultured MVECs, which were opposite to the effects induced by PES1 overexpression in MVECs. In vitro and in vivo experiments showed that high glucose concentration-induced increases in vascular paracellular permeability declined after MVECs were treated by β-HB or by knockdown of Pes1. In contrast, increases in vascular permeability were induced by overexpression of Pes1, which were suppressed by coadministration of β-HB in cultured endothelial cells. Similarly declines in vascular permeability were found by Pes1 knockdown in diabetic mice. Mechanistically, β-HB decreased PES1-facilitated ubiquitination of VE-cadherin. The KD suppressed the diabetes-induced increase in PES1, which may result in vascular hyperpermeability through ubiquitination of VE-cadherin in type 2 diabetic mice.

Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus.

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.