Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function:implication of miRNAs and microvesicles

Background Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis.We hypothesized that pericytes,a group of pluripotent cells that maintain vascular integrity and tension,are protective against sepsis via regulating vascular reactivity and permeability.Methods We conducted a series of in vivo experiments using wild-type(WT),platelet-derived growth factor receptor-β(PDGFR-β)-Cre+mT/mG transgenic mice and Tie2-Cre+Cx43^(flox/flox)mice to examine the relative contribution of pericytes in sepsis,either induced by cecal ligation and puncture(CLP)or lipopolysaccharide(LPS)challenge.In a separate set of experiments with Sprague-Dawley(SD)rats,pericytes were depleted using CP-673451,a selective PDGFR-βinhibitor,at a dosage of 40 mg/(kg·d)for 7 consecutive days.Cultured pericytes,vascular endothelial cells(VECs)and vascular smooth muscle cells(VSMCs)were used for mechanistic investigations.The effects of pericytes and pericyte-derived microvesicles(PCMVs)and candidate miRNAs on vascular reactivity and barrier function were also examined.Results CLP and LPS induced severe injury/loss of pericytes,vascular hyporeactivity and leakage(P<0.05).Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization(P<0.05).Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels(P<0.05).Additionally,PCMVs transferred miR-145 and miR-132 to VSMCs and VECs,respectively,exerting a protective effect on vascular reactivity and barrier function after sepsis(P<0.05).miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2(Sphk2)/sphingosine-1-phosphate receptor(S1PR)1/phosphorylation of myosin light chain 20 pathway,whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways.Conclusions Pericytes are protective against sepsis through regulating vascular reactivity and barrier function.Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

Knockdown of fibrillin-1 suppresses retina-blood barrier dysfunction by inhibiting vascular endothelial apoptosis under diabetic conditions

AIM:To investigate the effects of fibrillin-1(FBN1)deletion on the integrity of retina-blood barrier function and the apoptosis of vascular endothelial cells under diabetic conditions.METHODS:Streptozotocin(STZ)-induced diabetic mice were used to simulate the diabetic conditions of diabetic retinopathy(DR)patients,and FBN1 expression was detected in retinas from STZ-diabetic mice and controls.In the Gene Expression Omnibus(GEO)database,the GSE60436 dataset was selected to analyze FBN1 expressions in fibrovascular membranes from DR patients.Using lentivirus to knock down FBN1 levels,vascular leakage and endothelial barrier integrity were detected by Evans blue vascular permeability assay,fluorescein fundus angiography(FFA)and immunofluorescence labeled with tight junction marker in vivo.High glucose-induced monkey retinal vascular endothelial cells(RF/6A)were used to investigate effects of FBN1 on the cells in vitro.The vascular endothelial barrier integrity and apoptosis were detected by trans-endothelial electrical resistance(TEER)assay and flow cytometry,respectively.RESULTS:FBN1 mRNA expression was increased in retinas of STZ-induced diabetic mice and fibrovascular membranes of DR patients(GSE60436 datasets)using RNA-seq approach.Besides,knocking down of FBN1 by lentivirus intravitreal injection significantly inhibited the vascular leakage compared to STZ-DR group by Evans blue vascular permeability assay and FFA detection.Expressions of tight junction markers in STZ-DR mouse retinas were lower than those in the control group,and knocking down of FBN1 increased the tight junction levels.In vitro,30 mmol/L glucose could significantly inhibit viability of RF/6A cells,and FBN1 mRNA expression was increased under 30 mmol/L glucose stimulation.Down-regulation of FBN1 reduced high glucose(HG)-stimulated retinal microvascular endothelial cell permeability,increased TEER,and inhibited RF/6A cell apoptosis in vitro.CONCLUSION:The expression level of FBN1 increases in retinas and vascular endothelial cells under diabetic conditions.Down-regulation of FBN1 protects the retina of early diabetic rats from retina-blood barrier damage,reduce vascular leakage,cell apoptosis,and maintain vascular endothelial cell barrier function.

Ink-structing the future of vascular tissue engineering:a review of the physiological bioink design

Three-dimensional(3D)printing and bioprinting have come into view for a plannable and standardizable generation of implantable tissue-engineered constructs that can substitute native tissues and organs.These tissue-engineered structures are intended to integrate with the patient’s body.Vascular tissue engineering(TE)is relevant in TE because it supports the sustained oxygenization and nutrition of all tissue-engineered constructs.Bioinks have a specific role,representingthenecessarymedium for printability and vascular cell growth.This review aims to understand the requirements for the design of vascular bioinks.First,an in-depth analysis of vascular cell interaction with their native environment must be gained.A physiological bioink suitable for a tissue-engineered vascular graft(TEVG)must not only ensure good printability but also induce cells to behave like in a native vascular vessel,including self-regenerative and growth functions.This review describes the general structure of vascular walls with wall-specific cell and extracellular matrix(ECM)components and biomechanical properties and functions.Furthermore,the physiological role of vascular ECM components for their interaction with vascular cells and the mode of interaction is introduced.Diverse currently available or imaginable bioinks are described from physiological matrix proteins to nonphysiologically occurring but natural chemical compounds useful for vascular bioprinting.The physiological performance of these bioinks is evaluated with regard to biomechanical properties postprinting,with a view to current animal studies of 3D printed vascular structures.Finally,the main challenges for further bioink development,suitable bioink components to create a self-assembly bioink concept,and future bioprinting strategies are outlined.These concepts are discussed in terms of their suitability to be part of a TEVG with a high potential for later clinical use.

Impact of High Sodium Diet on Neovascularization and Osseointegration around Titanium Implant:An in Vivo and in Vitro Study

Objective A high sodium(HS)diet is believed to affect bone metabolism processes.Clarifying its impact on osseointegration of titanium(Ti)implants holds significant implications for postoperative dietary management of implanted patients.Methods This investigation probed the impact of sodium ions(Na^(+))on neovascularization and osteogenesis around Ti implants in vivo,utilizing micro-computed tomography,hematoxylin and eosin staining,and immunohistochemical analyses.Concurrently,in vitro experiments assessed the effects of varied Na^(+)concentrations and exposure durations on human umbilical vein endothelial cells(HUVECs)and MC3T3-E1 cells.Results In vivo,increased dietary sodium(0.8%-6.0%)led to a substantial decline in CD34 positive HUVECs and new bone formation around Ti implants,alongside an increase in inflammatory cells.In vitro,an increase in Na^(+)concentration(140-150 mmol/L)adversely affected the proliferation,angiogenesis,and migration of HUVECs,especially with prolonged exposure.While MC3T3-E1 cells initially exhibited less susceptibility to high Na^(+)concentrations compared to HUVECs during short-term exposure,prolonged exposure to a HS environment progressively diminished their proliferation,differentiation,and osteogenic capabilities.Conclusion These findings suggest that HS diet had a negative effect on the early osseointegration of Ti implants by interfering with the process of postoperative vascularized bone regeneration.

Understanding and controlling the variables for stromal vascular fraction therapy

In regenerative medicine,the isolation of mesenchymal stromal cells(MSCs)from the adipose tissue’s stromal vascular fraction(SVF)is a critical area of study.Our review meticulously examines the isolation process of MSCs,starting with the extraction of adipose tissue.The choice of liposuction technique,anatomical site,and immediate processing are essential to maintain cell functionality.We delve into the intricacies of enzymatic digestion,emphasizing the fine-tuning of enzyme concentrations to maximize cell yield while preventing harm.The review then outlines the filtration and centrifugation techniques necessary for isolating a purified SVF,alongside cell viability assessments like flow cytometry,which are vital for confirming the efficacy of the isolated MSCs.We discuss the advantages and drawbacks of using autologous vs allogeneic SVF sources,touching upon immunocompatibility and logistical considerations,as well as the variability inherent in donor-derived cells.Anesthesia choices,the selection between hypo-dermic needles vs liposuction cannulas,and the role of adipose tissue lysers in achieving cellular dissociation are evaluated for their impact on SVF isolation.Centrifugation protocols are also analyzed for their part in ensuring the integrity of the SVF.The necessity for standardized MSC isolation protocols is highlighted,promoting reproducibility and successful clinical application.We encourage ongoing research to deepen the understanding of MSC biology and therapeutic action,aiming to further the field of regenerative medicine.The review concludes with a call for rigorous research,interdisciplinary collaboration,and strict adherence to ethical and regulatory standards to safeguard patient safety and optimize treatment outcomes with MSCs.

Identification and Validation of Vascular-Associated Biomarkers for the Prognosis and Potential Pathogenesis of Hypertension Using Comprehensive Bioinformatics Methods

Background: Hypertension, also known as increased blood pressure, is a phenomenon in which blood flows in blood vessels and causes persistently higher-than-normal pressure on the vessel wall. The identification of novel prognostic and pathogenesis biomarkers plays a key role in the management of hypertension. Methods: The GSE7483 and GSE75815 datasets from the gene expression omnibus (GEO) database were used to identify the genes associated with hypertension that were differentially expressed genes (DEGs). The functional role of the DEGs was elucidated by gene body (GO) enrichment analysis. In addition, we performed an immune infiltration assay and GSEA on the DEGs of hypertensive patients and verified the expression of novel DEGs in the blood of hypertensive patients by RT-qPCR. Results: A total of 267 DEGs were identified from the GEO database. GO analysis revealed that these genes were associated mainly with biological processes such as fibroblast proliferation, cell structural organization, extracellular matrix organization, vasculature development regulation, and angiogenesis. We identified five possible biomarkers, Ecm1, Sparc, Sphk1, Thbsl, and Mecp2, which correlate with vascular development and angiogenesis characteristic of hypertension by bioinformatics, and explored the clinical expression levels of these genes by RT-qPCR, and found that Sparc, Sphk1, and Thbs1 showed significant up-regulation, in agreement with the results of the bioinformatics analysis. Conclusion: Our study suggested that Sparc, Sphk1 and Thbs1 may be potential novel biomarkers for the diagnosis, treatment and prognosis of hypertension and that they are involved in the regulation of vascular development and angiogenesis in hypertension.

Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult,greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.

In situ forming injectable MSC-loaded GelMA hydrogels combined with PD for vascularized sweat gland regeneration

Dear Editor,Three dimensional(3D)bioprinted extracellular matrix(ECM)can be used to provide both biochemical and biophysical cues to direct mesenchymal stem cells(MSCs)differentiation,and then differentiated cells were isolated for implantation in vivo using surgical procedures.However,the reduced cell activity after cell isolation from 3D constructs and low cell retention in injured sites limit its application[1].Methacrylated gelatin(GelMA)hydrogel has the advantage of fast crosslinking,which could resemble complex architectures of tissue construct in vivo[2].Here,we adopted a noninvasive bioprinting procedure to imitate the regenerative microenvironment that could simultaneously direct the sweat gland(SG)and vascular differentiation from MSCs and ultimately promote the replacement of glandular tissue in situ(Fig.1a).

3D printed grafts with gradient structures for organized vascular regeneration

Synthetic vascular grafts suitable for small-diameter arteries(<6 mm) are in great need.However,there are still no commercially available small-diameter vascular grafts(SDVGs) in clinical practice due to thrombosis and stenosis after in vivo implantation.When designing SDVGs,many studies emphasized reendothelization but ignored the importance of reconstruction of the smooth muscle layer(SML).To facilitate rapid SML regeneration,a high-resolution 3D printing method was used to create a novel bilayer SDVG with structures and mechanical properties mimicking natural arteries.Bioinspired by the collagen alignment of SML,the inner layer of the grafts had larger pore sizes and high porosity to accelerate the infiltration of cells and their circumferential alignment,which could facilitate SML reconstruction for compliance restoration and spontaneous endothelialization.The outer layer was designed to induce fibroblast recruitment by low porosity and minor pore size and provide SDVG with sufficient mechanical strength.One month after implantation,the arteries regenerated by 3D-printed grafts exhibited better pulsatility than electrospun grafts,with a compliance(8.9%) approaching that of natural arteries(11.36%) and significantly higher than that of electrospun ones(1.9%).The 3D-printed vascular demonstrated a three-layer structure more closely resembling natural arteries while electrospun grafts showed incomplete endothelium and immature SML.Our study shows the importance of SML reconstruction during vascular graft regeneration and provides an effective strategy to reconstruct blood vessels through 3D-printed structures rapidly.

Reliability of Echocardiographic Pulmonary Vascular Resistance to Screen for the New Definition of Precapillary Pulmonary Hypertension in Uncorrected Secundum Atrial Septal Defect

Background and Objective:The most feared complication of uncorrected secundum Atrial Septal Defect(ASD)is pulmonary arterial hypertension(PAH).Pulmonary vascular resistance(PVR)is crucial in detecting precapil-lary pulmonary hypertension(PH)to guide the need for PAH-specific therapy.There is a change in the cut-off value of PVR according to the recently updated PH guideline.How echocardiographic PVR(PVRecho)correlates to PVR by right heart catheterization(RHC)(PVRcath)according to the new guidelines has not been known.The aim of this study is to determine the reliability of PVRecho in detecting PAH in Uncorrected Ostium Secundum ASD based on the current updated guideline and to help screen the high PVR group.Methods:429 ostium secun-dum ASD in the COngenital HeARt Disease in Adult and Pulmonary Hypertension(COHARD-PH)registry was divided into three groups according to the PVR.PVRecho was calculated using Abbas’Formula and compared the its gold standard,the PVRcath.The correlation between the two methods was analyzed.The Bland-Altman plot was used to analyze the agreement between the two methods.Receiver operating characteristics(ROC)analysis was used to determine the PVRecho cut-off value for high PVR.Results:The majority of the population(63.5%)had high PVR.Female gender dominated the study population(84%).PVR_(echo) was significantly correlated with PVRcath(r=0.6225,p<0.0001).Bland-Altman plot among all groups and in subgroups analysis showed a wide range of agreement.PVRecho underestimated PVRcath 5.124 WU.In subgroup analysis,PVRecho overestimated PVRcath 0.35 WU in those with PVR<2 WU.In the second and third groups,PVR_(echo) underestimated PVRcath 0.52 and 10.77 WU,respectively.Conclusion:PVRecho is reliable in predicting high PVR in uncorrected secun-dum ASD.However,there is a wide range of agreement.PVR_(echo) cut-off value of>1.62 WU showed good dis-criminatory power in determining high PVR.

CircPMS1 promotes proliferation of pulmonary artery smooth muscle cells,pulmonary microvascular endothelial cells,and pericytes under hypoxia

Background:Circular RNAs(circRNAs)have been recognized as significant regulators of pulmonary hypertension(PH);however,the differential expression and function of circRNAs in different vascular cells under hypoxia remain unknown.Here,we identified co-differentially expressed circRNAs and determined their putative roles in the proliferation of pulmonary artery smooth muscle cells(PASMCs),pulmonary microvascular endothelial cells(PMECs),and pericytes(PCs)under hypoxia.Methods:Whole transcriptome sequencing was performed to analyze the differential expression of circRNAs in three different vascular cell types.Bioinformatic analysis was used to predict their putative biological function.Quantitative real-time polymerase chain reaction,Cell Counting Kit-8,and EdU Cell Proliferation assays were carried out to determine the role of circular postmeiotic segregation 1(circPMS1)as well as its potential sponge mechanism in PASMCs,PMECs,and PCs.Results:PASMCs,PMECs,and PCs exhibited 16,99,and 31 differentially expressed circRNAs under hypoxia,respectively.CircPMS1 was upregulated in PASMCs,PMECs,and PCs under hypoxia and enhanced the proliferation of vascular cells.CircPMS1may upregulate DEP domain containing 1(DEPDC1)and RNA polymerase II subunit D expression by targeting microRNA-432-5p(miR-432-5p)in PASMCs,upregulate MAX interactor 1(MXI1)expression by targeting miR-433-3p in PMECs,and upregulate zinc finger AN1-type containing 5(ZFAND5)expression by targeting miR-3613-5p in PCs.Conclusions:Our results suggest that circPMS1 promotes cell proliferation through the miR-432-5p/DEPDC1 or miR-432-5p/POL2D axis in PASMCs,through the miR-433-3p/MXI1 axis in PMECs,and through the miR-3613-5p/ZFAND5 axis in PCs,which provides putative targets for the early diagnosis and treatment of PH.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

Retinal vascular morphological characteristics in diabetic retinopathy: an artificial intelligence study using a transfer learning system to analyze ultra-wide field images

AIM:To investigate the morphological characteristics of retinal vessels in patients with different severity of diabetic retinopathy(DR)and in patients with or without diabetic macular edema(DME).METHODS:The 239 eyes of DR patients and 100 eyes of healthy individuals were recruited for the study.The severity of DR patients was graded as mild,moderate and severe non-proliferative diabetic retinopathy(NPDR)according to the international clinical diabetic retinopathy(ICDR)disease severity scale classification,and retinal vascular morphology was quantitatively analyzed in ultra-wide field images using RU-net and transfer learning methods.The presence of DME was determined by optical coherence tomography(OCT),and differences in vascular morphological characteristics were compared between patients with and without DME.RESULTS:Retinal vessel segmentation using RU-net and transfer learning system had an accuracy of 99%and a Dice metric of 0.76.Compared with the healthy group,the DR group had smaller vessel angles(33.68±3.01 vs 37.78±1.60),smaller fractal dimension(Df)values(1.33±0.05 vs 1.41±0.03),less vessel density(1.12±0.44 vs 2.09±0.36)and fewer vascular branches(206.1±88.8 vs 396.5±91.3),all P<0.001.As the severity of DR increased,Df values decreased,P=0.031.No significant difference between the DME and non-DME groups were observed in vascular morphological characteristics.CONCLUSION:In this study,an artificial intelligence retinal vessel segmentation system is used with 99%accuracy,thus providing with relatively satisfactory performance in the evaluation of quantitative vascular morphology.DR patients have a tendency of vascular occlusion and dropout.The presence of DME does not compromise the integral retinal vascular pattern.

Vascular medicine in the 21st century:Embracing comprehensive vasculature evaluation and multidisciplinary treatment

The field of vascular medicine has undergone a profound transformation in the 21st century,transforming our approach to assessment and treatment.Athero-sclerosis,a complex inflammatory disease that affects medium and large arteries,presents a major challenge for researchers and healthcare professionals.This condition,characterized by arterial plaque formation and narrowing,poses sub-stantial challenges to vascular health at individual,national,and global scales.Its repercussions are far-reaching,with clinical outcomes including ischemic heart disease,ischemic stroke,and peripheral arterial disease—conditions with esca-lating global prevalence.Early detection of vascular changes caused by athero-sclerosis is crucial in preventing these conditions,reducing morbidity,and averting mortality.This article underscored the imperative of adopting a holistic approach to grappling with the intricacies,trajectories,and ramifications of atherosclerosis.It stresses the need for a thorough evaluation of the vasculature and the implementation of a multidisciplinary treatment approach.By consi-dering the entire vascular system,healthcare providers can explore avenues for prevention,early detection,and effective management of this condition,ultima-tely leading to improved patient outcomes.We discussed current practices and proposed new directions made possible by emerging diagnostic modalities and treatment strategies.Additionally,we considered healthcare expenditure,resour-ce allocation,and the transformative potential of new innovative treatments and technologies.

Mufangji tang ameliorates pulmonary arterial hypertension through improving vascular remodeling,inhibiting inflammatory response and oxidative stress,and inducing apoptosis

Background:Mufangji tang(MFJT)is composed of Ramulus Cinnamomi,Radix Ginseng,Cocculus orbiculatus(Linn.)DC.,and Gypsum.In clinical settings,MFJT has been effectively employed in addressing a range of respiratory disorders,notably including pulmonary arterial hypertension(PAH).However,the mechanism of action of MFJT on PAH remains unknown.Methods:In this study,a monocrotaline-induced PAH rat model was established and treated with MFJT.The therapeutic effects of MFJT on PAH rat model were evaluated.Network pharmacology was conducted to screen the possible targets for MFJT on PAH,and the molecular docking between the main active components and the core targets was carried out.The key targets identified from network pharmacology were tested.Results:Results showed significant therapeutic effects of MFJT on PAH rat model.Analysis of network pharmacology revealed several potential targets related to apoptosis,inflammation,oxidative stress,and vascular remodeling.Molecular docking showed that the key components were well docked with the core targets.Further experimental validation results that MFJT treatment induced apoptosis(downregulated Bcl-2 levels and upregulated Bax levels in lung tissue),inhibited inflammatory response and oxdative stress(decreased the levels of IL-1β,TNF-α,inducible NOS,and malondialdehyde,and increased the levels of endothelial nitric oxide synthase,nitric oxide,glutathione and superoxide dismutase),reduced the proliferation of pulmonary arterial smooth muscle cells(downregulated ET-1 andβ-catenin levels and ERK1/2 phosphorylation,increased GSK3βlevels).Conclusion:Our study revealed MFJT treatment could alleviate PAH in rats via induction of apoptosis,inhibition of inflammation and oxidative stress,and the prevention of vascular remodeling.

Ultrasound-assisted isolation:A new method to isolate stromal vascular fraction

Background:The stromal vascular fraction(SVF),a cluster of stem and progenitor cells isolated from adipose tissue,holds significant promise for application in regenerative medicine.However,the existing methods for SVF isolation are time-consuming and expensive.Thus,in this study,we explored a new method of SVF extrac-tion-ultrasound-assisted SVF isolation(USASI)-and compared the viability and characteristics of SVF isolated using different methods.Methods:SVF extraction methods using different combinations of ultrasound power,ultrasound time,collagenase dosage,and collagenase digestion time were compared with those of the control group(collagenase digestion method).The cell yield and vitality of the SVF were evaluated via cell counting and trypan blue staining.The cell components and immunophenotypes of freshly isolated SVF were analyzed using flow cytometry.The prolifer-ative capacity and differentiation potential of the SVF were also identified.Results:Ultrasonication at 95 W-20 kHz for 30 s followed by digestion with 0.15%collagenase for 30 min was identified as the most suitable parameter for the USASI method in isolating SVF,as recommended based on the evaluation of various tested conditions.The USASI method significantly reduced the collagenase dosage and shortened the digestion time.Compared to the collagenase digestion method,the USASI method had a higher cell yield and cell viability,with no adverse effects on cell components,proliferative capacity,or multipotential differentiation capacity.Conclusion:With reduced processing time,lower collagenase dosage,and increased cell yield without impairing the viability and characteristics of SVF,USASI holds the potential to emerge as a time-saving and cost-effective method for future clinical applications.

Detecting early changes in choroidal vascularity and thickness using optical coherence tomography in patients with corneal crosslinking for keratoconus

AIM:To investigate changes in choroidal thickness and vascularity in keratoconus patients treated with corneal crosslinking.METHODS:This study evaluated 28 eyes of 22 patients with keratoconus who underwent corneal crosslinking.The choroidal thicknesses were evaluated on enhanced depth imaging optical coherence tomography at the preoperative and postoperative 3d,1,and 3mo.Choroidal thickness in the four cardinal quadrants and the fovea were evaluated.The choroidal vascularity index was also calculated.RESULTS:There was no significant difference in central choroidal thickness between the preoperative and postoperative 3d,1mo(P>0.05).There was a significant increase in the 3mo(P=0.034)and a significant decrease in the horizontal choroidal vascularity index on the postoperative 3d(P=0.014),there was no statistically significant change in vertical axes and other visits in horizontal sections(P>0.05).CONCLUSION:This study sheds light on choroidal changes in postoperative corneal crosslinking for keratoconus.While it suggests the procedure’s relative safety for submacular choroid,more extensive research is necessary to confirm these findings and their clinical significance.

Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy

AIM:To investigate the role of connective tissue growth factor(CTGF)and vascular endothelial growth factor(VEGF)in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy(PDR)following intravitreal injection of conbercept.METHODS:This study included 72 PDR patients and 8 cataract patients as controls.PDR patients were divided into 3 groups according to the intervals of 3,5,and 7d between intravitreal conbercept(IVC,0.5 mg/0.05 mL)injection and pars plana vitrectomy(PPV)performed.Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay(ELISA).The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on the data,and the protein interaction network of 23 differential proteins was studied.RESULTS:Post-IVC,VEGF levels decreased and CTGF levels increased significantly in aqueous humor,with the CTGF/VEGF ratio rising significantly at all intervals.Liquid chromatography tandem mass spectrometry(LC-MS/MS)identified differentially expressed proteins between preand post-IVC samples.GO and KEGG analyses revealed involvement in immune response,stress response,complement and coagulation cascades,ferroptosis,and PPAR signaling pathways.PPI analysis highlighted key proteins like APOA1,C3,and transferrin(TF).ELISA assay confirmed the differential expression of proteins such as HBA1,SERPINA1,COL1A1,and ACTB,with significant changes in the IVC groups.CONCLUSION:The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels,thereby modifying the CTGF/VEGF ratio,and IVC significantly alters the protein profile in the aqueous humor of patients with PDR.Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response,stress response,and cellular metabolism.

Analysis of vascular thrombus and clinicopathological factors in prognosis of gastric cancer:A retrospective cohort study

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors in the world,and its prognosis is closely related to many factors.In recent years,the incidence of vascular thrombosis in patients with GC has gradually attracted increasing attention,and studies have shown that it may have a significant impact on the survival rate and prognosis of patients.However,the specific mechanism underlying the association between vascular thrombosis and the prognosis of patients with GC remains unclear.AIM To analyze the relationships between vascular cancer support and other clinicopathological factors and their influence on the prognosis of patients with GC.METHODS This study retrospectively analyzed the clinicopathological data of 621 patients with GC and divided them into a positive group and a negative group according to the presence or absence of a vascular thrombus.The difference in the 5-year cumulative survival rate between the two groups was compared,and the relationships between vascular cancer thrombus and other clinicopathological factors and their influence on the prognosis of patients with GC were analyzed.RESULTS Among 621 patients with GC,the incidence of vascular thrombi was 31.7%(197 patients).Binary logistic regression analysis revealed that the degree of tumor differentiation,depth of invasion,and extent of lymph node metastasis were independent influencing factors for the occurrence of vascular thrombi in GC patients(P<0.01).The trend of the χ^(2) test showed that the degree of differentiation,depth of invasion,and extent of lymph node metastasis were linearly correlated with the percentage of vascular thrombi in GC patients(P<0.01),and the correlation between lymph node metastasis and vascular thrombi was more significant(r=0.387).Univariate analysis revealed that the 5-year cumulative survival rate of the positive group was significantly lower than that of the negative group(46.7%vs 73.3%,P<0.01).Multivariate analysis revealed that age,tumor diameter,TNM stage,and vascular thrombus were independent risk factors for the prognosis of GC patients(all P<0.05).Further stratified analysis revealed that the 5-year cumulative survival rate of stage Ⅲ GC patients in the thrombolase-positive group was significantly lower than that in the thrombolase-negative group(36.1%vs 51.4%;P<0.05).CONCLUSION Vascular cancer status is an independent risk factor affecting the prognosis of patients with GC.The combination of vascular cancer suppositories and TNM staging can better judge the prognosis of patients with GC and guide more reasonable treatment.