TSP-1 Expression Research Articles

Epigenetic regulation of Nrf2-Mediated angiogenesis in diabetic foot ulcer progression: Role of histone deacetylases

Nuclear factor E2-related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates proangiogenic mediators, and antioxidant and detoxification enzymes. However, hitherto its regulation in the progression of DFU was poorly examined. The regulation of Nrf2 has been reported to be affected by various factors, including histone deacetylase (HDACs) and DNA methylation. The present study aimed to profile all classes of HDACs and correlate them with Nrf2 and angiogenic markers in the tissue biopsies of different grades of DFU patients (n = 20 in each grade). The gene expression profile of Nrf2 and its downstream targets, angiogenic markers, and all classes of HDACs were assessed using qPCR. Spearman's correlation was performed to analyze the correlation of HDACs with Nrf2 and its downstream targets along with angiogenic markers. We observed a progressive decrease in the gene expression of Nrf2 and angiogenic markers such as VEGF, HIF-1α, and SDF-1α and also an increase in the TSP-2 expression in different grades of DFU. In parallel, a significant downregulation of HDAC2/8 and SIRT1/2/4 has been observed in various grades of DFU subjects. On the other hand, HDAC1/3/4/11 and SIRT3/5/6/7 showed upregulation in different grades of DFU and the maximum increase was observed in Grade 3 patients. A significant negative correlation between Nrf2 and HDAC4, angiogenic markers, and HDAC4 suggested the pivotal role of the HDAC4-regulated Nrf2-mediated angiogenesis among DFU subjects. We have generated a first line of evidence on the epigenetic regulation of Nrf2 and its correlation with angiogenesis in the progression of diabetic foot ulcers.

Novel Role of Endothelial CD47 in the Regulation of Pathogenesis of Coronary Artery Disease

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with CVD. However, the role of endothelial cell-specific expression of CD47 in the pathogenesis and progression of atherosclerosis has not yet been fully defined. Kojima et.al. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelial cell-specific CD47 regulates efferocytosis in atherosclerosis. Methods: Endothelial cell-specific, tamoxifen-inducible CD47knockout (CD47iECKO) mice were injected with a single dose of AAV-mutant PCSK9 via the tail vein. Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in mice fed Western Diet for 12 weeks. Using single-cell RNA sequencing and in vitro efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in an atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to a significant plaque reduction in the aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from athero-control and athero-CD47iECKO mice identified 10 major cell types from the integrated data: endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells (B cells, T cells, macrophage), erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. After Western Diet intake, in the CD47iECKO group, a subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, resident aortic macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using an in vitro efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to BAY11-7082 (NF-κB inhibitor) to stimulate the apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single cell level during atherogenesis and provides a deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for this cardiovascular disease. NIH, AHA. 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.

High glucose environment induces NEDD4 deficiency that impairs angiogenesis and diabetic wound healing

BackgroundHealing of diabetic wounds, characterized by impaired angiogenesis, remains a clinical challenge. E3 ligase have been identified as potential therapeutic targets of wound healing. ObjectiveWe assessed the role of E3 ligase NEDD4 in the context of angiogenesis and diabetic wound healing. MethodsThe mRNA expression levels of NEDD4, TSP1 and VEGF were determined by real-time PCR. Western blotting was used to evaluate the protein expression of NEDD4, TSP1 and VEGF. The ubiquitination of TSP1 was evaluated by immunoprecipitation. MTT assay, wound healing assay and tube formation assay were performed to assess the proliferation, migration and angiogenic functions of endothelial cells. The epigenetic modification in the promoter of NEDD4 was confirmed using BSP assay and ChIP-qPCR assay. The role of NEDD4 in wound healing was further verified in diabetic mouse model. ResultsNEDD4 promotes proliferation, migration and tube formation of endothelial cells. It binds to and ubiquitinates TSP1, which lead to TSP1 degradation and thus increased VEGF expression. The inhibitory effect of NEDD4 silencing on the angiogenesis ability of endothelial cells can be restored by TSP1 knockdown. NEDD4 is reduced in diabetic patients, which may due to hypermethylation of NEDD4 promoter mediated via DNMT1 under high glucose condition. Furthermore, inhibition of NEDD4 represses wound healing in diabetic mouse model. ConclusionNEDD4 might promote angiogenesis and wound healing by inhibiting TSP1 via ubiquitination in diabetic patients.

PIAS1 impedes vascular endothelial injury and atherosclerotic plaque formation in diabetes by blocking the RUNX3/TSP-1 axis.

The protein PIAS1 functions as a type of ubiquitin-protease, which is known to play an important regulatory role in various diseases, including cardiovascular diseases and cancers. Its mechanism of action primarily revolves around regulating the transcription, translation, and modification of target proteins. This study investigates role and mechanism of PIAS1 in the RUNX3/TSP-1 axis and confirms its therapeutic effects on diabetes-related complications in animal models. A diabetic vascular injury was induced in human umbilical vein endothelial cells (HUVECs) by stimulation with H2O2 and advanced glycation end product (AGE), and a streptozotocin (STZ)-induced mouse model of diabetes was constructed, followed by detection of endogenous PIAS1 expression and SUMOylation level of RUNX3. Effects of PIAS1 concerning RUNX3 and TSP-1 on the HUVEC apoptosis and inflammation were evaluated using the ectopic expression experiments. Down-regulated PIAS1 expression and SUMOylation level of RUNX3 were identified in the H2O2- and AGE-induced HUVEC model of diabetic vascular injury and STZ-induced mouse models of diabetes. PIAS1 promoted the SUMOylation of RUNX3 at the K148 site of RUNX3. PIAS1-mediated SUMOylation of RUNX3 reduced RUNX3 transactivation activity, weakened the binding of RUNX3 to the promoter region of TSP-1, and caused downregulation of TSP-1 expression. PIASI decreased the expression of TSP-1 by inhibiting H2O2- and AGE-induced RUNX3 de-SUMOylation, thereby arresting the inflammatory response and apoptosis of HUVECs. Besides, PIAS1 reduced vascular endothelial injury and atherosclerotic plaque formation in mouse models of diabetes by inhibiting the RUNX3/TSP-1 axis. Our study proved that PIAS1 suppressed vascular endothelial injury and atherosclerotic plaque formation in mouse models of diabetes via the RUNX3/TSP-1 axis.

SYNJ1 rescues motor functions in hereditary and sporadic Parkinson's disease mice by upregulating TSP-1 expression

This study aimed to explore the role of SYNJ1 in Parkinson's disease (PD) and its potential as a neuroprotective factor. We found that SYNJ1 was decreased in the SN and striatum of hSNCA*A53T-Tg and MPTP-induced mice compared to normal mice, associated with motor dysfunction, increased α-synuclein and decreased tyrosine hydroxylase. To investigate its neuroprotective effects, SYNJ1 expression was upregulated in the striatum of mice through injection of the rAdV-Synj1 virus into the striatum, which resulted in the rescue of behavioral deficiencies and amelioration of pathological changes. Subsequently, transcriptomic sequencing, bioinformatics analysis and qPCR were conducted in SH-SY5Y cells following SYNJ1 gene knockdown to identify its downstream pathways, which revealed decreased expression of TSP-1 involving extracellular matrix pathways. The virtual protein-protein docking further suggested a potential interaction between the SYNJ1 and TSP-1 proteins. This was followed by the identification of a SYNJ1-dependent TSP-1 expression model in two PD models. The coimmunoprecipitation experiment verified that the interaction between SYNJ1 and TSP-1 was attenuated in 11-month-old hSNCA*A53T-Tg mice compared to normal controls. Our findings suggest that overexpression of SYNJ1 may protect hSNCA*A53T-Tg and MPTP-induced mice by upregulating TSP-1 expression, which is involved in the extracellular matrix pathways. This suggests that SYNJ1 could be a potential therapeutic target for PD, though more research is needed to understand its mechanism.

Extracellular status of thrombospondin-2 in type 2 diabetes mellitus and utility as a biomarker in the determination of early diabetic kidney disease

ObjectiveThrombospondin-2 (TSP-2) is a multifunctional matricellular glycoprotein correlated with glucose homeostasis, insulin sensitivity, and estimated glomerular filtration rate. Investigation of the association of TSP-2 with type 2 diabetes mellitus (T2DM) and the potential diagnostic value of serum TSP-2 for detecting early diabetic kidney disease (DKD) is needed.Research design and methodsAn enzyme-linked immunosorbent assay was used for detection serum TSP-2 levels in 494 Chinese T2DM subjects. The protein expression of TSP-2 in the kidney and other tissues were tested by western blotting.ResultsSerum TSP-2 levels in T2DM subjects were significantly higher than in healthy individuals. Serum TSP-2 correlated positively with triglycerides, serum uric acid, creatinine, platelets, and urinary albumin-to-creatinine ratio (UACR), but negatively with estimated glomerular filtration rate, after adjusting for age, sex, and T2DM duration. Logistic regression analysis demonstrated an independent association between serum TSP-2 and early DKD. Furthermore, the high UACR identified at risk of early DKD increased significantly from 0.78 (95%CI 0.73–0.83) to 0.82 (95%CI 0.77–0.86, p < 0.001) when added to a clinical model consisting of TSP-2 and age. In db/db mice, serum TSP-2 levels were elevated. TSP-2 expression was markedly increased in the kidney tissue compared with that in db/m and m/m mice. Furthermore, serum TSP-2 expression correlated well with UACR in mice.ConclusionsTSP-2 is a novel glycoprotein associated with early DKD in patients with T2DM. The paradoxical increase of serum TSP-2 in T2DM individuals may be due to a compensatory response to chronic inflammatory and renal vascular endothelial growth, warranting further investigation.

Abstract 338: Novel Role Of Endothelial Cd47 In The Regulation Of Pathogenesis Of Atherosclerosis

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with cerebrovascular disease. However, the role of endothelial cell-specific CD47 in the pathogenesis of atherosclerosis has not yet been defined. Kojima et.al . described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelialCD47 regulates efferocytosis in atherosclerosis. Methods: Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in atherosclerotic mice. Using single-cell RNA sequencing and efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to significant plaque reduction in aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from atherosclerotic mice identified 10 major cell types: endothelial cells, VSMCs, fibroblasts, B cells, T cells, macrophage, erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. In athero-CD47iECKO group, subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to NF-κB inhibitor, BAY11-7082 to stimulate apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single-cell level during atherogenesis and provides deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for cardiovascular disease.

Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

Combination of Radix Astragali and Safflower Promotes Angiogenesis in Rats with Ischemic Stroke via Silencing PTGS2.

Promotion of angiogenesis and restoration of the blood flow in the ischemic penumbra is an effective treatment for patients with ischemic stroke (IS). Radix astragali-safflower (AS), a classic herbal pair for accelerating blood circulation and dispersing blood stasis, has been used for thousands of years to treat patients with IS in China. Even so, the mechanism of the treatment of IS by AS is still undecipherable. In the current study, network pharmacology was firstly employed to unveil the mechanism of AS in treating IS, which showed that AS might promote angiogenesis associated with PTGS2 silence. Middle cerebral artery occlusion/reperfusion (MCAO/R) model rats were then used as the experimental animals to verify the prediction result. The experimental results revealed that treatment with AS improved the cerebral infarct volume, neurological damage, and cerebral histopathological damage; inhibited cell apoptosis; increased the contents of PDGF-BB, EPO, and TGF-β1; and reduced the levels of PF4, Ang-2, and TIMP-1 in serum. Immunohistochemical staining demonstrated that the expression of PTGS2 was dramatically increased in the hippocampus and cerebral cortex of rats with MCAO/R, and this trend was reversed by the treatment of AS. Immunofluorescent staining expressed that AS reversed the down-regulation of VEGF and further promoted the expression of CD31, which indicated that AS promoted angiogenesis in MCAO/R rats. The abnormal protein or mRNA expression of PTGS2, PGI2, bFGF, TSP-1, and VEGF in the penumbra were transposed by AS or Celecoxib (an inhibitor of PTGS2). In conclusion, the protective mechanism of AS for IS promoted angiogenesis and was involved with PTGS2 silence.

Deficiency of thrombospondin-2 alleviates intimal hyperplasia in mice by modulating vascular smooth muscle cell proliferation and migration.

Thrombospondin-2 (Tsp2), a glycoprotein in the extracellular matrix, plays a critical role in the maintenance of vascular homeostasis. However, its role in the pathogenesis of cardiovascular disorders such as intimal hyperplasia is not fully elucidated. This study, therefore, aims to explore the effect of Tsp2 on intimal hyperplasia and its associated underlying mechanisms. Intimal hyperplasia (IH) was established using a modified wire-mediated femoral artery injury model. Immunofluorescence and qPCR identified upregulated Tsp2 expression in the injured femoral artery compared with the uninjured femoral artery. Similarly, TSP2 expression was also increased in human samples from the atherosclerotic femoral artery and colocalized with vascular smooth muscle cells (VSMCs). Compared with the wild-type littermates, Tsp2 knockout mice displayed a mitigated IH in the injured femoral artery, as demonstrated by a decreased neointimal area and intimal/median ratio. Primary mouse VSMCs were cultured to explore the mechanism by which Tsp2 influenced IH in vitro. PDGF-stimulated VSMCs presented an elevated Tsp2 expression and enhanced migration and proliferation. However, Tsp2 knockdown by siRNA blocked the increased migration and proliferation of VSMCs. Further analysis identified an association between Notch3 and IH when the intracellular domain of Notch3 (Nicd3) was upregulated in PDGF-stimulated VSMCs and femoral arteries with IH in human tissues. Along with the overexpression and downregulation of Tsp2, the Nicd3 expression was also up and downregulated accordingly. Tsp2 was associated with IH and may serve as a therapeutic target for IH. Downregulation of Tsp2 could mitigate the progression of IH by modulating the proliferation and migration of VSMCs.

The Mechanism of miR-21-5p/TSP-1-Mediating Exercise on the Function of Endothelial Progenitor Cells in Aged Rats.

(1) Background: The declined function of peripheral circulating endothelial progenitor cells (EPCs) in aging individuals resulted in decreased endothelial cell regeneration and vascular endothelial function. Improving EPCs function in aging individuals plays an important role in preventing cardiovascular diseases. (2) Methods: Thirty aged (18-month-old) male Sprague-Dawley rats were randomly divided into control and exercise groups. An aerobic exercise intervention was performed 5 days/week for 8 weeks. EPCs functions, miR-21-5p, and TSP-1 expressions were detected after the intervention. The senescence rate, proliferation, and migration of EPCs were examined after overexpression of miR-21-5p and inhibition of TSP-1 expression. (3) Results: The senescence rate, proliferation, and migration of EPCs in exercise groups were significantly improved after exercise intervention. The miR-21-5p expression was increased and the TSP-1 mRNA expression was decreased in the EPCs after the intervention. miR-21-5p overexpression can improve EPCs function and inhibit TSP-1 expression but has no effect on senescence rate. Inhibition of TSP-1 expression could improve the function and reduce the senescence rate. (4) Conclusions: Our results indicate that long-term aerobic exercise can improve the functions of EPCs in aging individuals by downregulating TSP-1 expression via miR-21-5p, which reveals the mechanism of exercise in improving cardiovascular function.

PRSS2 remodels the tumor microenvironment via repression of Tsp1 to stimulate tumor growth and progression

The progression of cancer from localized to metastatic disease is the primary cause of morbidity and mortality. The interplay between the tumor and its microenvironment is the key driver in this process of tumor progression. In order for tumors to progress and metastasize they must reprogram the cells that make up the microenvironment to promote tumor growth and suppress endogenous defense systems, such as the immune and inflammatory response. We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression. Here, we identify a novel tumor-mediated mechanism that represses the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a hitherto undescribed activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1. Based on the ability of PRSS2 to reprogram the tumor microenvironment, this discovery could lead to the development of therapeutic agents that are indication agnostic.

Astrocytic ERK/STAT1 Signaling Contributes to Maintenance of Stress-Related Visceral Hypersensitivity in Rats

The rostral anterior cingulate cortex (rACC) has been found to be an important brain region in mediating visceral hypersensitivity. However, the underlying mechanisms remain unclear. This study aimed to explore the role of astrocytes in the maintenance of visceral hypersensitivity induced by chronic water avoidance stress (WAS) as well as the potential signaling pathway that activates astrocytes in the rACC. We found that ACC-reactive astrogliosis resulted in the overexpression of c-fos, TSP-1, and BDNF in stress-related visceral hypersensitivity rats. Visceral hypersensitivity was reversed by pharmacological inhibition of astrocytic activation after WAS, as were the overexpression of c-fos, TSP-1 and BDNF. Activation of the astrocytic Gi-pathway increased the visceral sensitivity and expression of c-fos, TSP-1, and BDNF. Visceral hypersensitivity was also ameliorated by the pharmacological inhibition of ERK and STAT1 phosphorylation after WAS. Furthermore, inhibition of the ERK-STAT1 cascade reduced astrocytic activation. These findings suggest that astrocytic ERK/STAT1 signaling in the rACC contributes to the maintenance of stress-related visceral hypersensitivity. PerspectiveVisceral hypersensitivity is a key factor in the pathophysiology of irritable bowel syndrome. This study highlights the important role of astrocytic ERK/STAT1 signaling in activating astrocytes in the rostral anterior cingulate cortex, which contributes to visceral hypersensitivity.

CD47 Binding on Vascular Endothelial Cells Inhibits IL-17-Mediated Leukocyte Adhesion.

To address the conflicting role of thrombospondin (TSP)-1 reported in acute and chronic pathologies, this study investigated the role of TSP-1 in regulating leukocyte recruitment and regulation of VCAM-1 expression using mouse models of uveitis. The spontaneously increased VCAM-1 expression and leukocyte adhesion in retinas of TSP-1-deficient mice suggested a TSP-1-mediated regulation of VCAM-1 expression. In a chronic uveitis model, induced by immunizing wild-type mice with specific interphotoreceptor retinoid-binding protein (IRBP) peptide, topically applied TSP-1-derived CD47-binding peptide significantly reduced the clinical disease course and retinal leukocyte adhesion as compared to the control peptide-treated group. In contrast, in LPS-mediated acute uveitis, TSP-1 deficiency significantly reduced the retinal leukocyte adhesion. The results of our in vitro study, using vascular endothelial cell (EC) cultures, demonstrate that unlike TNF-α, VCAM-1 expression induced by IL-17 is associated with a reduced expression of endogenous TSP-1. Such reduced endogenous TSP-1 expression in IL-17-stimulated ECs helps limit the CD36-mediated increased VCAM-1 expression, while favoring CD47-mediated inhibition of VCAM-1 expression and leukocyte adhesion. Thus, our study identifies TSP-1:CD47 interaction as a molecular pathway that modulates IL-17-mediated VCAM-1 expression, contributing to its anti-inflammatory effect in chronic inflammatory conditions.

LPS‐induced endotoxemia promotes blood‐brain barrier permeability via TSP1‐dependent TGFβ1 activation

IntroductionDuring disease states, the blood‐brain barrier (BBB) can become permeable due to endothelial injury or dysregulation of tight junction proteins. Endotoxemia, which induces systemic inflammation, causes BBB dysfunction and neuroinflammation. We have shown that transforming growth factor beta 1 (TGFβ1) is upregulated during systemic inflammation and promotes BBB permeability by disrupting claudin‐5 and occludin. Therefore, we hypothesize that TGFβ1 is upregulated during endotoxemia and induces BBB permeability by disrupting tight junction proteins.MethodsC57Bl/6 mice were administered 3 mg/kg of LPS and monitored for neurological decline. In a second set of mice 6 hours after LPS administration, an antagonist to TGFβ1 activation, LSKL, or control, SLLK, were injected (1 mg/kg/ip). Mice were euthanized 24 hours after LPS injection and tissue was collected. In a subset of animals, 3 hours prior to euthanasia, Evans Blue was injected (8%/ip) to assess BBB function. TGFβ1, claudin‐5, occludin, IL‐1β, IL6 and TNFα expression was assessed by RTPCR, immunoblotting and immunofluorescence. In vitro studies using mouse brain endothelial cells (bEnd.3 cells) were treated with LPS (10 μg/ml) or rTSP1 (1 μg/ml) to activate TGFβ1. TGFβ1, TSP1, claudin‐5 and occludin expression was assessed by immunofluorescence or RTPCR. Barrier function was determined in bEnd.3 cell monolayers using TEER or 10‐kDa FITC‐dextran diffusion. Data were analyzed using GraphPad Prism with results presented as mean +/‐ SEM. Differences between two groups were compared using Student’s t‐test and differences between three groups were determined using ANOVA.ResultsTGFβ1 expression was increased in cortex and cerebellum of LPS‐treated mice. This was associated with decreased claudin‐5 and occludin mRNA expression and immunofluorescence staining. Treatment of bEnd.3 cells with LPS increased TGFβ1 and TSP1 expression, decreased TEER, and increased FITC‐dextran diffusion. Supplementation of bEnd.3 cells with rTSP1 led to decreased claudin‐5 and occludin mRNA expression, decreased TEER, and increased FITC‐dextran diffusion across the transwell. LSKL treatment in LPS‐treated mice reduced sickness behavior, reduced Evan’s blue penetrance into the brain, restored tight junction expression and alleviated neuroinflammation when compared to SLLK‐treated mice.ConclusionsStrategies to target TGFβ1 signaling may prove effective at alleviating endotoxin‐induced BBB dysfunction.

Research on Mechanism of Hypoxia-Inducible Factor-1α Gene Carried with Albumin Nanoparticles in Regulating Tumor Angiogenesis in Human Papillomavirus Through Induction of PI3K/AKT Signal Pathway

The aim of this study was assessing the mechanism of HIF-1α gene carried with albumin nanoparticles in regulating the tumor angiogenesis in HPV through induction of PI3K/AKT signal pathway. 40 of Wistar rats were divided into blank set, HIF-1α set, agonist set and inhibitor set randomly. There were 10 rats in every set. The gene expression of HIF-1α, presentation rate of CD31 and SMA, protein expression of VEGF and TSP-1, and presentation of factors related with PI3K/AKT signal pathway were all studied. HIF-1α gene carried with albumin nanoparticles was prepared successfully, and the gene expression of HIF-1α in HIF-1α set was highest. The second highest expression was in the inhibitor set, while the lowest was in the blank and agonist sets. There were notably-visible tan particles in the tumor tissue from the blank, HIF-1α and agonist sets, and they were reduced notably in the inhibitor set. The presentation rates for CD31 and SMA in the tumor tissues from the blank, HIF-1α and agonist sets were highest, while the rate was reduced in the inhibitor set. The action on VEGF protein expression was similar, and there was positive correlation between abnormal activation of HIF-1α and tumor angiogenesis in HPV. The PI3k/AKT signal transduction pathway was activated by HIF-1α and PI3k and AKI were phosphorylated. The HIF-1α was prompted to enter into cell nucleus and interact with element with low oxygen effect so as to increase the presentation of VEGF, CD31 and SMA. The angiogenesis in the HPV was also prompted, but the action was eliminated by inhibitor from the PI3K/AKT pathway.

Cordycepin enhances anti-tumor immunity in colon cancer by inhibiting phagocytosis immune checkpoint CD47 expression

Cordycepin, also known as 3′-deoxyadenosine, is an extract from Cordyceps militaris, which has been reported as an anti-inflammation and anti-tumor substance without toxicity. However, the pharmacological mechanism of Cordycepin on tumor immunity under its anti-tumor effect has not yet been elucidated. Herein, we investigated Cordycepin’s anti-tumor effect on colon cancer both in vitro and in vivo. Our results show that Cordycepin can inhibit growth, migration, and promoted apoptosis of CT26 cells in a dose-dependent manner. Cordycepin suppressed the growth of colon cancer in mouse subcutaneous tumor model by modulating tumor immune microenvironment where CD4+ T, CD8+ T, M1 type macrophages, NK cells were up-regulated. Further investigations revealed that Cordycepin inhibited phagocytosis immune checkpoint CD47 protein expression by reducing BNIP3 expression. In addition, Cordycepin also inhibited the expression of TSP1 in tumor cells and Jurkat cells, which may reduce the binding of TSP1 to CD47, thereby reducing T cell apoptosis and allowing more T cells to infiltrate into tumors. And in vitro co-culture experiments proved that Cordycepin could enhance the phagocytosis of CT26 cells by macrophages. These results explained the underlying mechanism of the anti-tumor immunity of Cordycepin. In conclusion, our results identify a novel mechanism by which Cordycepin inhibits phagocytosis immune checkpoint CD47 in tumor cells to promote tumor cells phagocytosis of macrophages. Cordycepin may be able to serve as a more effective immunotherapeutic drug against colon cancer.

Effects of plasma lipopolysaccharide changes on platelet release of vascular endothelial growth factor and thromobospondin-1 in patients with cirrhotic portal hypertension after TIPS procedure

Objective: To investigate the effects of plasma lipopolysaccharide (LPS) concentration changes on platelet release of vascular endothelial growth factor (VEGF) and thrombospondin (TSP)-1 in patients with decompensated cirrhotic portal hypertension after transjugular intrahepatic portosystemic shunt (TIPS) procedure. Methods: 169 cases with cirrhotic portal hypertension were enrolled, of which 81 cases received TIPS treatment. LPS, VEGF, and TSP-1 concentrations with different Child-Pugh class in peripheral blood plasma of patients were measured. After pre-incubation of normal human platelets with different concentrations of LPS and stimulated by collagen in vitro, platelet PAC-1 expression rate, VEGF, and TSP-1 concentrations were detected. PAC-1 expression rate and the concentrations of LPS, VEGF and TSP-1 in peripheral blood plasma of patients before and after TIPS procedure were detected. The relationship between plasma LPS, VEGF and TSP-1 concentrations and Child-Pugh score changes in patients after TIPS procedure was analyzed. Statistical analysis was performed by t-test, one-way ANOVA or Pearson's rho according to different data. Results: Plasma LPS and TSP-1 concentrations were significantly higher in Child-Pugh class C patients than class A and B, but the concentration of plasma VEGF was significantly lower than class A and B (P < 0.01). In vitro experiments showed that concentration of LPS, TSP-1, and platelet PAC-1 expression rate was higher in the supernatant, but the difference in the concentration of VEGF in the supernatant was not statistically significant. Portal vein pressure and platelet activation were significantly decreased (P < 0.01) in patients after TIPS procedure. Portal venous pressure, platelet activation, plasma LPS, and TSP-1 levels were significantly decreased continuously, while VEGF levels were significantly increased continuously after TIPS procedure. Plasma LPS concentration was positively correlated with TSP-1 concentration (r = 0.506, P < 0.001), and negatively correlated with VEGF concentration (r = -0.167, P = 0.010). Child-Pugh score change range was negatively correlated with change range of plasma VEGF concentration (r = -0.297, P = 0.016), and positively correlated with change range of plasma TSP-1 concentration (r = 0.145, P = 0.031) after TIPS. Conclusion: Portal venous pressure gradient, plasma LPS concentration and corresponding platelet activation was decreased in cirrhotic portal hypertension after TIPS procedure, and with TSP-1 reduction and VEGF elevation it is possible to reduce the liver function injury caused by portal venous shunt.

Differential action of pro-angiogenic and anti-angiogenic components of Danhong injection in ischemic vascular disease or tumor models

ObjectiveWe investigate the chemical basis and mechanism of angiogenesis regulation by a multicomponent Chinese medicine Danhong injection (DHI).MethodsDHI was fractionated and screened for angiogenesis activities by in vitro tube formation and migration assays. The composition of DHI components was determined by UPLC. The effects of the main active monomers on angiogenesis-related gene and protein expression in endothelial cells were determined by qPCR and Western blotting analyses. Mouse hind limb ischemia and tumor implant models were used to verify the angiogenesis effects in vivo by Laser Doppler and bioluminescent imaging, respectively.ResultsTwo distinct chemical components, one promoting (pro-angiogenic, PAC) and the other inhibiting (anti-angiogenic, AAC) angiogenesis, were identified in DHI. PAC enhanced angiogenesis and improved recovery of ischemic limb perfusion while AAC reduced Lewis lung carcinoma growth in vivo in VEGFR-2-Luc mice. Among the PAC or AAC monomers, caffeic acid and rosmarinic acid upregulated TSP1 expression and downregulated KDR and PECAM expression. Caffeic acid and rosmarinic acid significantly decreased while protocatechuic aldehyde increased CXCR4 expression, which are consistent with their differential effects on EC migration.ConclusionsDHI is capable of bi-directional regulation of angiogenesis in disease-specific manner. The pro-angiogenesis activity of DHI promotes the repair of ischemic vascular injury, whereas the anti-angiogenesis activity inhibits tumor growth. The active pro- and anti-angiogenesis activities are composed of unique chemical combinations that differentially regulate angiogenesis-related gene networks.

Effects of cyanocobalamin and its combination with morphine on neuropathic rats and the relationship between these effects and thrombospondin-4 expression.

BackgroundThrombospondin-4 (TSP4) upregulates in the spinal cord following peripheral nerve injury and contributes to the development of neuropathic pain (NP). We investigated the effects of cyanocobalamin alone or in combination with morphine on pain and the relationship between these effects and spinal TSP4 expression in neuropathic rats.MethodsNP was induced by chronic constriction injury (CCI) of the sciatic nerve. Cyanocobalamin (5 and 10 mg/kg/day) was administered 15 days before CCI and then for 4 and 14 postoperative days. Morphine (2.5 and 5 mg/kg/day) was administered only post-CCI. Combination treatment included cyanocobalamin and morphine, 10 and 5 mg/kg/day, respectively. All drugs were administered intraperitoneally. Nociceptive thresholds were detected by esthesiometer, analgesia meter, and plantar test, and TSP4 expression was assessed by western blotting and fluorescence immunohistochemistry.ResultsCCI decreased nociceptive thresholds in all tests and induced TSP4 expression on the 4th postoperative day. The decrease in nociceptive thresholds persisted except for the plantar test, and the increased TSP4 expression reversed on the 14th postoperative day. Cyanocobalamin and low-dose morphine alone did not produce any antinociceptive effects. High-dose morphine improved the decreased nociceptive thresholds in the esthesiometer when administered alone but combined with cyanocobalamin in all tests. Cyanocobalamin and morphine significantly induced TSP4 expression when administered alone in both doses for 4 or 14 days. However, this increase was less when the two drugs are combined.ConclusionsThe combination of cyanocobalamin and morphine is more effective in antinociception and partially decreased the induced TSP4 expression compared to the use of either drug alone.