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Purpose To investigate the histological efficacy of ranibizumab and zoledronic acid in an experimentally induced endometriosis model as compared with danazol, buserelin acetate and dienogest. Methods Endometrial implants were introduced in 52 female Wistar albino rats, which were then randomly divided into six groups. The animals were, respectively, given dienogest, danazol, buserelin acetate, zoledronic acid, ranibizumab and 0.9% NaCl. After 4 weeks, the volumes and histopathological properties of the implants were evaluated and the implants were excised completely at the third laparotomy. A histopathological scoring system was used to evaluate the preservation of epithelia. Endometrial explants were evaluated immunohistochemically. Results Among the groups, the histological score was significantly lower in the zoledronic acid and ranibizumab groups compared with the controls (p < 0.001). There were no significant differences regarding ellipsoidal volume levels between groups (p > 0.05). However, there was a statistically significant difference regarding cell numbers according to the degree of Bcl-2, NF-kappa B, and CD31 staining (p < 0.001). There was no statistically significant difference in Bcl-2, CD31, or NF-kappa B staining in the binary comparisons between the other groups (p > 0.05). For Bcl-2 staining, the staining rate of the group treated with zoledronic acid was significantly lower compared with the dienogest and danazol groups (p < 0.05). The staining rates of CD31 and NF-kappa B were significantly lower in the zoledronic acid and ranibizumab groups compared with the controls (p < 0.05). Conclusion According to these results, zoledronic acid and ranibizumab may be putative candidates for the treatment of endometriosis., Technological and Scientific Research Council of Turkey (TUBITAK) [213S002], This study was funded by The Technological and Scientific Research Council of Turkey (TUBITAK) (213S002).

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Successful implantation involves a high degree of development of spiral arteries, combined with high proliferative activity, which ensures the formation of a healthy placenta, full uterine-placental circulation, and the birth of a healthy child. The placenta is the unique organ of the biological monitoring, the mirror of pregnancy. Identification of placental markers of miscarriage is a promising direction for preventing reproductive losses. The aim of the work is to identify the markers of miscarriage and premature labor in the structures of the chorion and placenta. Materials and methods. The main group included tissue samples of the 22 chorions and 64 placentas after termination of current pregnancy from women with a history of reproductive losses. The control group included tissue samples of the 20 chorions after artificial abortion and 20 placentas after physiological pregnancy and birth. The placenta was examined according to the protocol (form No. 013-1/0). The expressions of VEGF, CD31/PECAM1, CD105/Endoglin/TGFβ 1/3 Receptor, Bcl-2α Ab-1, TNF-α, CD45/T200/LCA, CD56/NCAM1 were studied in the structures of chorion and placenta by immunohistochemistry. Results. Based on histological and immuno-histochemical study of chorion and placenta samples in women with reproductive history and termination of the current pregnancy, it was established that embryo-endometrial dysfunction is the cause of miscarriage in the first trimester, and inflammation is the precondition of preterm birth; markers of miscarriage and premature labor in the structures of the chorion and placenta have been identified. Conclusions. The markers of miscarriage are pathomorphological changes in endometrium and chorion combined with high expression of TNF-α and NK-CD56, low expression of CD31/PECAM1, negative expression of VEGF to indicate a violation of cytotrophoblast invasions. The markers of inflammation and premature labor are structural and functional changes of placenta in combination with moderate expression of TNF-α in syncytium, high expression of NK-CD56 in villous stroma, high expression of CD45/T200/LCA in the decidual membrane.

BACKGROUND TAS-102 is effective against unresectable advanced or recurrent colorectal and gastric cancer. However, its effect on oral squamous cell carcinoma (OSCC) is still unknown. Here, we tried to clarify the possible effect of TAS-102 against angiogenesis and proliferation of human OSCC cells. MATERIALS AND METHODS 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration assay and mice xenograft models were used to determine the effect of TAS-102 on growth and migration of OSCC. The activity of phosphorylated nuclear factor kappa light-chain-enhancer of activated B-cells (NF-κB) (p-p65) in cells was detected by immunocytochemistry. The expression of p-AKT serine/threonine kinase 1 (p-AKT), p-p65, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2) and CD31 in mouse tumors were detected by immunohistochemistry. RESULTS TAS-102 significantly inhibited growth and migration of OSCC both in vitro and in vivo. It suppressed the activity of NF-κB in cells. TAS-102 down-regulated the expression of p-AKT, VEGF, FGF2 and CD31, which was associated with reduced vascularization of HSC2 tumor lesions. CONCLUSION These findings suggest that TAS-102 might inhibit angiogenesis and proliferation of OSCC cells.

Kaposi sarcoma is a malignant vascular tumor consisting of multiple clinical subtypes and varying histopathologic patterns. We report a case of a 53-year-old African-American male with HIV/AIDS who presented multiple times with skin nodules, pain, and edema in his lower extremities, secondary to recurrent Kaposi sarcoma. The patient was treated with two courses of liposomal doxorubicin with improvement, but his symptoms recurred a third time. A biopsy specimen of one of the nodules showed prominent neoplastic cells of epithelioid morphology, some with clear cell change, appearing to form rudimentary vessels in the superficial dermis. Further inspection of the deeper dermis revealed more classic findings of Kaposi sarcoma, including admixed spindle cells, poorly-defined vessels, scattered apoptotic bodies, entrapped collagen bundles, and extravasated erythrocytes. Both the epithelioid and classic portions of the neoplasm stained positive for CD31 and human herpesvirus 8, supporting a diagnosis of Kaposi sarcoma. Prior to this case, the epithelioid variant of Kaposi sarcoma has only been reported twice in the literature. Recognizing this rare histopathologic variant of Kaposi sarcoma among its other histopathologic patterns may assist in accurate and expedient diagnosis of this well-recognized disease. This article is protected by copyright. All rights reserved.

Epithelioid hemangioma (EH) is a benign vascular lesion, typically consisting of small vascular channels lined by epithelioid endothelial cells and associated with a dense lymphocytic infiltrate with eosinophils. Here, we report a rare case of EH involving large arteries. The patient presented with a nine-month history of an asymptomatic nodule on the forehead, which was thought to be an epidermal inclusion cyst. Skin biopsy revealed large arteries with clusters of epithelioid cells in the vascular walls and lumen. Scattered eosinophils were noted in the walls. Adjacent areas showed groups of small-caliber vessels lined by prominent endothelial cells and associated with a dense lymphoid infiltrate with eosinophils. No significant cytologic atypia was noted. Given the presence of the classic small vessel involvement, along with CD31 reactivity for the epithelioid cells in the large vessels, the findings are classified as EH involving large arteries, which is an uncommon subtype. There have only been a handful of such cases reported in the literature. This article is protected by copyright. All rights reserved.

Composite hemangioendothelioma (CHE) is considered a borderline malignant vascular tumor defined by an admixture of distinct vascular neoplastic components. A 21-year-old female is presented herein with a 1 cm painless mandibular vestibular mass of less than a year duration. The infiltrating tumor was characterized by dilated vascular channels lined by endothelial cells with bland ovoid or round nuclei exhibiting, occasionally, hobnail/matchstick-like arrangement. Intravascular cell proliferations with hyaline globular deposits were also present. Additionally, lobular spindle and epithelioid cell aggregates, as well as slit-like spaces exhibiting a retiform or angiosarcomatous morphology were observed. Intracytoplasmic signet-ring or lipoblast-like vacuolization was also noted. Mitotic activity was exceptionally rare. Vascular spaces and the stroma featured lymphocytes and plasma cells. Neoplastic cells were positive for CD31, CD34, D2-40 and ERG, negative for CAMTA1 and synaptophysin, while type IV collagen highlighted the plasmalemma of most vessels and hyaline globules. Fluorescence in situ hybridization revealed gene rearrangements in both YAP1 and MAML2 genes, in keeping with a YAP1-MAML2 fusion. Whole exome sequencing (WES) identified three missense mutations FLT1 [p.R1016G], PIK3CA [p.H1047L], and C11orf42 [p.A304P] and a mitochondrial frameshift insertion MT-ND4 [c.1107_1108insC; p.P370fs]. These WES results suggest that FLT1 and/or PIK3CA variants may contribute to tumor growth/transformation while the MT-ND4 variant may relate to proliferation, angiogenesis and/or inhibition of apoptosis.

INTRODUCTION The objective of this study was to assess the diagnostic utility of CD10 in the differential diagnosis of grade 1-endometrial endometrioid carcinoma (G1-EEC) and the metaplastic changes associated with the endometrial glandular and stromal breakdown (EGBD) on liquid-based cytological (LBC) samples. METHODS (1) The type and distribution of CD10-positive cells in EGBD and G1-EEC patients were evaluated. (2) Based on the results from (1), histological and cytological specimens were double-immunostained with CD31 and CD10 to confirm whether CD10-positive tubular-canalicular material found in (1) was represented by fine threads of endometrial-type fibrovascular stroma. (3) Based on the results from (2), additional immunostaining of histological specimens was performed for CD146 and αSMA as markers of perivascular cells. RESULTS (1) CD10 positive cells showed two main patterns of expression: cytoplasmic immunoreactivity in the form of dense brown granules in EGBD and tubular-canalicular branching patterns in G1-EEC. (2) The tubular-canalicular material observed in cytological specimens of G1-EEC samples co-expressed CD10 and CD31, and was interpreted as representing fine threads of endometrial fibrovascular stroma in the corresponding histological samples. Conversely, metaplastic changes in EGBD cases, only a few CD31-positive signals were found inside the condensed stromal clusters with CD10-positive. (3) Cells surrounding the CD31-positive vascular endothelial cells expressed CD146 and αSMA; moreover, some of the thin CD10-positive fibrous stromal strands also co-expressed αSMA. CONCLUSIONS CD10 is a very useful immunomarker for distinguishing between G1-EEC and the metaplastic changes of EGBD in LBC samples.

Epithelial to mesenchymal transition (EMT) is an important mechanism of invasion in cutaneous squamous cell carcinomas (cSCCs) and has been found to be enhanced in tumors originated from actinic keratosis with transformation limited to the basal epithelial layer -differentiated pathway-, compared to cases with invasion subsequent to complete epidermal transformation -classical pathway-. Several microRNAs and proteins can contribute to EMT modulation in cSCCs. MicroRNA21 and microRNA31 are involved in posttranscriptional regulation of protein expression and could play a relevant role in EMT and cSCC progression. Throughout the EMT process upregulation of matrix metalloproteinases (MMPs) enhances invasiveness and MMP-1 and MMP-3 contribute to local invasion, angiogenesis and metastasis in cSCCs. Additionally, cSCC development is associated with PTEN loss and NF-kappa B, NOTCH-1 and p63 activation. The aim of this work is to identify differences in the expression of those molecules between both pathways of cSCCs development. Eight tissue microarrays from 80 consecutive cSCCs were analyzed using LNA-based miRNA in situ hybridization for miRNA21 and miRNA31 evaluation, and immunohistochemistry for MMP-1, MMP-3, PTEN, NOTCH-1, NF-kappa B, p63 and CD31. Significantly higher expression of miRNA31 (p < 0.0001) and MMP-1 (p = 0.0072) and angiogenesis (p = 0.0199) were found in the differentiated pathway, whereas PTEN loss (p = 0.0430) was more marked in the classical pathway. No significant differences were found for the other markers. Our findings support a contribution of miRNA31 and MMP-1 in the differentiated pathway, associated to EMT and increased microvascularization. The greater PTEN loss in the classical pathway indicate that its relevance in cSCC is not EMT-related.

Amelotin (AMTN) is a protein that is expressed during the maturation of dental enamel and has important role in enamel hydroxyapatite mineralization. However, it is not well understood whether AMTN has a strong mineral-promoting ability in bone. In this study, the effect of AMTN on bone healing was investigated using mice calvarial defect model in vivo, and the expression of bone marker genes and cell proliferation were investigated to clarify the role of AMTN in bone mineralization using mouse osteogenic cells (MC3T3-E1) in vitro. Collagen membranes, with or without recombinant human (rh) AMTN, were applied to calvarial defects created on the parietal bones of C57BL/6N mice. Microcomputed tomography and histological observation revealed that the defect largely filled with mineralized tissue by the rhAMTN-containing membrane in eight weeks. Moreover, CD31 positive cells were observed in the newly formed mineralized tissue and around the rhAMTN-containing membrane. In the presence of rhAMTN, the expression of the Spp1 gene in MC3T3-E1 cells significantly increased within ten days in an osteoinductive medium. Moreover, rhAMTN significantly enhanced MC3T3-E1 cell proliferation. These findings indicate that AMTN positively influences bone repair by promoting hydroxyapatite mineralization.

Skin burns are one of the most common injuries associated with increased morbidity and mortality, especially in the children and the elderlies. Severe burns, especially, result in a systemic immune and inflammatory response, which may reflect in multiple organ insufficiency, and a fast and effective local restorative process is essential for functionality recovering, as well as for interrupting the generalized systemic response. We have aimed here to assess the effect of different wound dressings in what it regards the morphology and clinical restoration after a skin burn. On a rat animal model, we have evaluated the macroscopic and histopathological features of controlled third degree skin burns in control animals versus treatments with local dressings of silver sulfadiazine (SDA) cream, simple gel (G), gel + silver nanoparticles (AgNPs) (G+NPS), gel + exosomes (G+EXO) and gel + AgNPs + exosomes (Gel+NPS+EXO), at 14 days and, respectively, 21 days after the lesion. Tissue fragments were harvested and processed for histopathology and immunohistochemistry. Immunofluorescence was utilized to evaluate the maturity of underlaying granulation tissue based on double stainings for smooth muscle actin (SMA) and cluster of differentiation 31 (CD31). Our study showed variability in what it regards the vessel density and immunoexpression of SMA between the treatments, and image analysis revealed that most SMA reduction and blood vessel density reduction in the maturing granulation tissue occurred for the G+NPS and G+NPS+EXO treatments. A complete re-epithelization was also observed for the G+NPS+EXO treatment. Overall, our results show that improved topic treatments promote faster re-epithelization and reparation of the dermis after skin burn lesions, providing thus an avenue for new treatments that aim both local recuperation and systemic infection prevention.

The distinction between mucinous carcinomas (MCs) and mucocele-like lesions (MLLs), particularly those containing detached epithelial fragments, can be problematic in the limited samples afforded by breast core needle biopsies (CNBs). Neovascularization of mucin has been proposed as a criterion to distinguish MC from MLL, but its value in helping to categorize mucin-producing breast lesions in CNB has not been previously investigated. To address this, we evaluated mucin neovascularization on hematoxylin and eosin (HE)-stained sections of 140 CNB containing mucin-producing breast lesions including 52 MC, 17 mucin-producing ductal carcinoma in situ (mDCIS), and 71 MLL. In 116 cases with sufficient remaining material (42 MC, 16 mDCIS, and 58 MLL), we also assessed mucin neovascularization on CD31 immunostains. On HE-stained sections, neovascularization of mucin, defined as delicate, thin-walled microvessels in mucin, and unassociated with fibrous septae, was identified significantly more frequently in MC than in MLL (69.2% vs. 14.1%; P=0.0001). The difference in the frequency of mucin neovascularization between MC and MLL was even greater on CD31 immunostains (97.6% vs. 13.8%, P0.00001). The sensitivity, specificity, positive predictive value, and negative predictive value of mucin neovascularization for categorizing a lesion as MC were 69.2%, 85.8%, 78.3%, and 79.2%, respectively, for HE-stained sections and 97.6%, 86.2%, 83.7%, and 98.0%, respectively, for CD31 immunostains. We conclude that mucin neovascularization is significantly more common in MC than in MLL in breast CNB on HE-stained sections and particularly on CD31 immunostains and may be a valuable adjunct in distinguishing between MC and MLL in problematic cases.

This study describes the histomorphology and immunohistochemical profile of 9 cases of feline piloleiomyosarcoma. Cats ranged in age from 7 to 16 years (mean 10), and tumors were 7 to 24 mm in diameter (mean 15). Tumors were composed of fusiform cells that were haphazardly arranged or in variably sized interwoven bundles. Neoplastic cells had eosinophilic and fibrillar cytoplasm, and elongated blunt-ended nuclei. Entrapment of hair follicles and absence of vascular components support an origin from the smooth muscle cells of the arrector pili. Additional findings included bizarre nuclei and giant cells (7/9 cases), atypical mitoses (7/9 cases), ulceration (3/9 cases), and intratumoral necrosis (6/9 cases). Neoplastic cells expressed calponin, desmin, α-smooth muscle actin, and vimentin, but not CD18, CD31, cytokeratins, glial fibrillary acidic protein, neuron-specific enolase, Melan A, p63, or S-100 protein. Surgical excision was curative in 6/9 cases, with local recurrence in 2/9 cases and metastasis to local lymph nodes in 1/9 case. Clinical outcome was influenced by mitotic count, infiltration of subcutaneous tissue, and intensity of nuclear immunolabeling for p53.

Background and Objective: Angiogenesis is the most important repair process of tissues subjected to ischemic injury. The present study aims to investigate whether the pro-angiogenic effect of Qiliqiangxin prescription (QL) is mediated through miR-21 signaling. Methods: Cardiac microvascular endothelial cells (CMECs) were isolated and cultured from 2-3 weeks old SD rats by the method of planting myocardium tissues. The purity was identified by CD31 immunofluorescence staining. CMECs were then cultured under 1% O2 hypoxia or normoxia condition for 24h in the presence or absence of QL pretreatment (QL, 0.5mg/ml, 24h). The mimics and inhibitors of miR-21 were transfected into CMECs. miR-21, HIF-1α, and VEGF expressions of CMECs were then detected by qRT-PCR and/or Western blot. The proliferation, migration, and tube formation functions of CMECs were assessed using the BrdU assay, wound healing test, and tube formation assay, respectively. Results: The results showed that compared with the control group, hypoxia significantly upregulated the expression of miR-21 and impaired CMECs proliferation, migration, and tube formation functions. Compared with the hypoxia group, QL further upregulated miR-21, HIF-1α, and VEGF expressions, and improved cell proliferation, migration, and tube formation of hypoxic CMECs. These effects of QL were abolished by a knockdown of miR-21. Conversely, treatment with miR-21 mimics further enhanced QL induced changes in hypoxic CMECs. Conclusions: Results indicate that the pro-angiogenesis effects of QL on hypoxic CMECs are mediated by activating miR-21 and its downstream HIF-1α/VEGF pathway possibly.

Background Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA positron emission tomography/computed tomography has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. Methods We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium (CM) derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the CM. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived CM using a tube formation assay that mimics angiogenesis. Results Immunohistochemistry of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in 4 of 33 prostate cancer patients (12.1%). We also found that the 10,000g pellet fraction of the LNCaP-derived CM containing PSMA-positive membranes, such as microvesicles transformed HUVECs "PSMA-negative" into "PSMA-positive." Furthermore, treatment of HUVECs with the 10,000g pellet fraction of the LNCaP-derived CM significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. Conclusions Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues.

Aim. To create a personalized cell-populated small-diameter vascular prosthesis in a pulsating bioreactor.Methods. Tubular grafts were made by electrospinning from mixtures of biodegradable polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(εcaprolactone) (PCL). The inner surface is modified with fibrin. Tubular scaffolds were colonized with cultured colony-forming endothelial cells and grown under static conditions for 2 days. Then, the cell-populated prostheses continued to be cultivated for 5 days in a pulsating bioreactor system with a final shear stress of 2.85 dynes/cm².Results. The advantages of the cultivation of cell-populated vascular prostheses in a pulsating bioreactor have been revealed. The selected mode of cultivation of cellpopulated vascular prostheses under conditions of a pulsating flow with a shear stress of 2.85 dynes/cm² did not have a damaging effect on the integrity of the endothelial monolayer. Moving unidirectional mechanical stimuli of chaotic orientation fibers of F-actin changed to a predominant orientation in the direction of flow, and also increased the expression of F-actin, Talin focal adhesion protein, and specific endothelial markers CD309, CD31, vWF.Conclusion. The creation of a personalized cell-populated small-diameter vascular prosthesis with a functional endothelial monolayer is possible due to the use of autologous endothelial cells, autologous fibrin, and cultivation under conditions of a pulsating flow.

Here we examined the effect of nicotine on angiogenesis in the brain after intracerebral hemorrhage (ICH), as angiogenesis is considered to provide beneficial effects on brain tissues during recovery from injury after stroke. Nicotine was administered to C57BL/6 mice suffering from collagenase-induced ICH in the striatum, either by inclusion in drinking water or by daily intraperitoneal injection. Nicotine administration by both routes enhanced angiogenesis within the hematoma-affected regions, as revealed by increased CD31-immunopositive area at 7 and 14 d after ICH. Double immunofluorescence histochemistry against CD31 and proliferating cell nuclear antigen revealed that nicotine increased the number of newly generated vascular endothelial cells within the hematoma. In spite of enhanced angiogenesis, nicotine did not worsen vascular permeability after ICH, as assessed by Evans Blue extravasation. These effects of nicotine were accompanied by an increased number of surviving neurons in the hematoma at 7 d after ICH. Unexpectedly, nicotine did not increase expression of vascular endothelial growth factor mRNA in the brain and did not enhance recruitment of endothelial progenitor cells from the bone marrow. These results suggest that nicotine enhances angiogenesis in the brain after ICH, via mechanisms distinct from those involved in its action on angiogenesis in peripheral tissues.

BACKGROUND: Constructing tissue-engineered kidneys using decellularized kidney scaffolds (DKS) has attracted widespread attention as it is expected to be the key to solving the shortage of donor kidneys. However, thrombosis and the host inflammatory response are unfavorable factors that hider the re-endothelialization and vascularization of the decellularized scaffolds. METHODS: Heparin was immobilized into the DKS using the method of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) activation. Fourier-transform infrared (FTIR) spectra were used to verify the heparinization of DKS. Human umbilical vein endothelial cells (HUVECs) were seeded and cultured in the DKS, then the sliced scaffolds were transplanted subcutaneously into nude mouse. Scanning electron microscopy and a series of histochemical stains including hematoxylin and eosin (H&E), elastic Verhöeff-Van Gieson (EVG), Sirius red, Masson’s trichrome, and toluidine blue (TB) staining were used for morphological characterization. The qRT-PCR analysis, immunohistochemistry (IHC), and immunofluorescence (IF) staining were used to determine the expression of related molecular markers. RESULTS: The rat DKS completely retained the extracellular matrix and heparinized modification. The H&E staining results showed there were more HUVECs covering the internal surfaces of tubular structures in the HEP-DKS group compared with the DKS group. The IF analysis results revealed that CD31, Ki67, and CD206 had higher positive rates in HUVECs in the HEP-DKS group compared to the DKS group. Both groups of scaffolds showed blood vessel formation via H&E staining, and there were more blood vessels in the HEP-DKS group compared with the native DKS group (P

Purpose The involvement of the circulating endothelium-derived microparticles (EMPs) and the endothelial progenitor cells (EPCs) has been shown in the pathogenesis of coronary artery disease (CAD). The current study aimed to explore whether the Friesinger index is associated with the levels of the apoptotic CD144+/CD31+/annexin V+ ​EMPs and the number of endothelial colony-forming units of progenitor cells in patients undergoing coronary angiography. Patients and methods Fifty-seven patients with a median age of 62 years (range: 48–84 years) were enrolled. Quantification of the apoptotic CD144+/CD31+/annexin V+ EMPs was performed by flow cytometry. The number of endothelial colony-forming units defined by CFU-Hill was assessed by cell culture. Results There was a positive correlation between the Friesinger index and the circulating levels of the apoptotic CD144+/CD31+/annexin V+ EMPs (rho=0.817, p Conclusions Moderate or severe CAD is associated with increased levels of apoptotic EMPs and reduced EPC colony-forming capacity, increasing the occurrence of endothelial injuries.

Objectives Irritation fibroma in the oral cavity causes atrophy or squamous epithelium thickening with respect to external injury-associated factors. However, ulcers do not occur in most cases. This study aimed to elucidate the mechanism by which ulcers do not form, focusing on the vascular network in the mucosal epithelium of irritation fibroma. Methods Immunostaining was performed using an enzyme antibody method with primary antibodies against CD31 and Ki-67 in 17 cases of irritation fibroma in the buccal mucosa. One section was taken at three points from the margin and three points from just above the lesion for measurement. The number of blood vessels in the superficial and deep lamina propria at the measurement site were determined, and the area per blood vessel was measured. Results The number and area of blood vessels in the superficial lamina propria just below the lesion epithelium were smaller than those in the margin. No difference was observed in the number and area of blood vessels in the deep lamina propria between the margins and lesions. Conclusions Our results suggest that the vascular network in the deep lamina propria is maintained and compensates for the nutrient supply to the covering epithelium.

Thymic output and homeostatic mature cell proliferation both influence T cell repopulation following depletional induction, though the relative contribution of each and their association with recipient age have not been well studied. We investigated the repopulating T cell kinetics in kidney transplant recipients who underwent alemtuzumab induction followed by belatacept/rapamycin-based immunosuppression over 36-month posttransplantation. We focused specifically on the correlation between repopulating T cell subsets and the age of patients. Substantial homeostatic Ki67-expressing T cell proliferation was seen posttransplantation. A repertoire enriched for naive T (TNaive ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaive cells and advancing age. A relationship between age and persistence of effector memory cells was shown. We assessed thymic output and found an increase in the frequency of recent thymic emigrants (RTEs, CD4+ CD31+ ) at 12-month posttransplantation. Patients under 30 years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55 years of age pre- and posttransplantation. IL-7 and autologous mature dendritic cells (mDCs) induced CD57- cell proliferation. In contrast, mDCs, but not IL-7, induced CD57+ cell proliferation. This study establishes the relationship between age and thymic output during T cell homeostatic repopulation after alemtuzumab induction. Trial Registration: ClinicalTrials.gov - NCT00565773.

OBJECTIVE We examined the hypothesis that antibiotic treatment with or without gonadotropin releasing hormone agonist (GnRHa) may decrease intrauterine infection with consequent decrease in tissue inflammation, cell proliferation and angiogenesis in human endometriosis. STUDY DESIGN This is a prospective non-randomized observational study. Endometrial/endometriotic samples were collected during surgery from 53 women with endometriosis and 47 control women who were treated with levofloxacin (LVFX, 500 mg, once per os) or GnRHa (1.88 mg/IM for 3 months) before surgery. Endometrial samples were analyzed by broad-range polymerase-chain reaction (PCR) amplification of bacteria targeting V5-V6 region of 16S rRNA gene. Immunohistochemical analysis was performed using antibodies against CD138 (Syndecan-1, a marker of plasma cells), CD68 (marker of macrophages), Ki-67 (cell proliferation marker), and CD31 (vascular cells marker). RESULTS 16S rDNA metagenome assay indicated that treatment with either of LVFX or GnRHa + LVFX significantly decreased some components of major bacterial genera comparing to untreated group. In women with endometriosis, treatment with either of LVFX or GnRHa + LVFX significantly decreased Gardnerella, Prevotella, Acidibactor, Atopobium, Megasphaera, and Bradyrhizobium (p

An increasing number of epithelioid vascular lesions, in particular tumors from the benign and low-grade end of the spectrum, have been characterized by recurrent gene fusions. As a result, the detection of these molecular markers have improved the classification of diagnostically challenging cases. However, despite the significant progress, there are occasional lesions that do not fit in known histologic or molecular groups. Herein, we present five such unclassified epithelioid vascular lesions, which occurred in the bone and showed a distinct morphology composed of alternating vasoformative and solid growth and mild to moderate nuclear pleomorphism. The variegated morphologic appearance resembled that of composite hemangioendothelioma, being distinct from both epithelioid hemangioma and epithelioid hemangioendothelioma, and consistently showed cytologic atypia. Due to their unusual morphologic appearance and negative molecular work-up, targeted transcriptome sequencing was performed in two cases showing the presence of NFATC2 fusions with either EWSR1 or FUS genes. Three additional bone tumors with EWSR1 gene rearrangements were identified by FISH screening of a large cohort of 45 fusion-negative epithelioid vascular neoplasms, one fused to NFATC2 while two others to NFATC1. There were three females and two males, with a wide age range at presentation, mean of 44 years. The lesions occurred in the pelvis, maxillary sinus, and humerus. Two patients presented with polyostotic disease, both located in the pelvic bones. Two patients had available follow-up, one developed two local recurrences in the humerus over a 15-year period, while the other showed no recurrence 4 years subsequent to an en-bloc resection. Tumors were positive for CD31 and ERG, while negative for EMA, CK, synaptophysin, and chromogranin. FISH confirmed this abnormality in all cases, none of them being associated with gene amplifications. Further studies are needed to establish the pathogenetic relationship of this rare molecular subset with other epithelioid vascular tumors and to determine its clinical behavior.

Summary A 10-year-old spayed female Border Collie developed a ductal adenocarcinoma in the spleen. Clinically, the spleen was enlarged and a small liver nodule was present but there were no other abnormalities. Most of the splenic parenchyma was diffusely infiltrated by variably shaped atypical neoplastic cells that formed small clusters or larger nests, arranged as duct or duct-like structures within a fibrous matrix. There was acinar differentiation in a few portions of the tumour with a sheet-like solid growth pattern and occasional squamous metaplasia or exocrine acinus-like structures. Mitotic figures were frequent. Neoplastic cells with ductal differentiation were diffusely immunoreactive for AE1/AE3, CAM5.2 and CK7 cytokeratins but negative for CK20, while cells with acinar differentiation were immunolabelled only for AE1/AE3 cytokeratins and were also immunopositive for mucin-1 and trypsin. A few regions of tumour with ductal or acinar differentiation were immunopositive for pancreatic lipase. All neoplastic cells were negative for mucin-2, vimentin, smooth muscle actin, chromogranin A, CD31, hepatocyte paraffin 1 and thyroglobulin antigens. Because of the formation of exocrine acinus-like structures and an immunolabelling pattern consistent with exocrine pancreas tissue, an adenocarcinoma of ectopic exocrine pancreas within the spleen was diagnosed.

© 2021 Wiley Periodicals LLCObjectives: Unpredictability with the final volume and viability of the graft are the major concerns in fat grafting. An experimental study was conducted to increase graft retention using photobiomodulation (PBM) with polychromatic light in near-infrared region (600–1200 nm) by utilizing its stimulatory effects on angiogenesis, neovascularization, adipocyte viability, and anti-inflammatory properties. Methods: A total of 24 rats were divided into four groups (n = 6) according to the applied polychromatic light protocol to the recipient site (none, before fat transfer, after fat transfer, and combined). In all groups, inguinal fat pad was excised, measured for volume and weight, and transferred to the dorsum of the rat. At the end of the experiment, fat grafts were harvested from the recipient site for volume and weight measurements, histological, and immunohistochemical evaluation. Results: Intergroup comparison revealed that fat graft retention regarding weight and volume, was significantly superior in Group IV (p = 0.049 and p = 0.043, respectively), which polychromatic light was applied both before and after transfer of the graft. Hematoxylin–eosin and Masson's trichrome stained sections showed absence of necrosis, fibrosis, inflammation, cyst formation, and increased vascularization of both inner and outer zones of the grafts in Group IV. Also, immunohistochemical staining scores for perilipin (indicator for adipocyte viability), CD31 and VEGF (indicators for angiogenesis and neovascularization) were significantly higher (p < 0.001). Ki67 scores were significantly lower in this group because of anti-inflammatory environment (p < 0.001). Conclusions: Application of PBM to the recipient site before and after fat transfer improved outcomes in rats at 56 day after fat grafting by means of volume retention, increased neovascularization and adipocyte viability and reduced necrosis, fibrosis and inflammation.

This study was aimed at investigating whether Elabela (ELA) gene therapy can promote angiogenesis in the treatment of myocardial infarction (MI). The fusion expression plasmid pAAV‐3 × Flag/ELA‐32 was successfully constructed using molecular cloning technique. The model of acute MI was established by ligating the left anterior descending coronary artery in mice. Adeno‐associated virus serotype 9 (AAV9) was injected into the surrounding myocardium and tail vein immediately after the model was established. AAV was injected again from the tail vein one week later. Compared with the MI+PBS (control) group, the serum N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) concentration, and the values of left ventricular end‐diastolic diameter (LVDd) and left ventricular end‐systolic diameter (LVDs) of the MI+AAV‐ELA (gene therapy) group were significantly decreased, while the value of left ventricular ejection fraction was significantly increased at 2 and 4 weeks after operation. Compared with the control group, the expression of CD105 and vWF and the percentage of CD31‐ and Ki67–co‐positive cells were significantly increased in the gene therapy group. Moreover, the expressions of apelin peptide jejunum (APJ) receptor, vascular endothelial growth factor (VEGF), VEGFR2, Jagged1 and Notch3 in the heart tissue around the infarction were up‐regulated in mice with gene therapy. The results suggest that ELA activates VEFG/VEGFR2 and Jagged1/Notch3 pathways through APJ to promote angiogenesis after myocardial infarction. ELA gene therapy may be used in the treatment of ischaemic cardiomyopathy in future.

BACKGROUND Endothelial microparticles (EMPs) act as early biomarkers of endothelial activation and damage. No studies have investigated EMPs in preterm-born individuals. METHODS Sixty-three preterm-born children and 52 children born full-term (controls) were studied. Circulating CD62E(+), CD144(+), and CD31(+)/CD42b(-) EMPs were measured in preterm-born children compared to controls; possible associations with cardiovascular risk factors and endothelial function parameters were also assessed. RESULTS Circulating CD62E(+), CD144(+), and CD31(+)/CD42b(-) EMPs were significantly higher in preterm-born children compared to controls (p = 0.003, p

The effects of chemotherapy on the tumor microenvironment remain poorly investigated, although these are crucial for the drug delivery and anti-tumor activity and can modify physiological behavior of cancer cells. Optical coherence angiography and phosphorescence lifetime spectroscopy are promising optical methods to characterize both tumor vasculature and its oxygenation, respectively. In this study, we used these methods to explore the influence of irinotecan, a widely used chemotherapeutic agent, on tumor angiogenesis and oxygen content in a mouse model of colorectal cancer in vivo . For the first time, it was shown that irinotecan at conventional dosing reduces tumor oxygenation and blood perfusion via antiangiogenic mechanism of action. Immunohistochemistry for hypoxia and for the endothelial cell marker CD31 verified the in vivo findings. These data show the prospects of the optical techniques to monitor tumor microenvironment in the course of chemotherapy, which can help to optimize and individualize the treatment.

Pluripotent stem cells (PSCs) are a promising source of endothelial cells (ECs) for the treatment of cardiovascular diseases. Since clinical application of embryo stem cells (ESCs) involves issues of medical ethics and risk of immune rejection, induced pluripotent stem cells (iPSCs) will facilitate cell transplantation therapy for the cardiovascular diseases. Swine is identified as an ideal large-animal model for human, because of its similar organ size and physiological characteristics. However, there are very few studies on EC differentiation of porcine iPSCs (piPSCs). In recent study, we provided an efficient protocol to differentiate piPSCs into ECs with the purity of 19.76% CD31 positive cells within 16 days. Passaging of these cells yielded a nearly pure population, which also expressed other endothelial markers such as CD144, eNOS and vWF. Besides, these cells exhibited functions of ECs such as uptake of low-density lipoprotein and formation of tubes in vitro or blood vessels in vivo. Our study successfully obtained ECs from piPSCs via a feeder- and serum-free monolayer system and demonstrated their angiogenic function in vivo and in vitro. piPSC-ECs derivation is not only potential for the autologous cell transplantation and cardiovascular drug screening, but also for the mechanistic studies on EC differentiation and endothelial dysfunction.

The platelet endothelial cell adhesion molecule-1 (PECAM-1) or CD31 has been involved in regulation of T-cell tolerance, activation, survival and homing in mice cells. However, there is limited knowledge about the expression pattern and role of this molecule in human T cells, particularly in conditions of chronic immune activation.We explored CD31 expression in T cell differentiation subsets of individuals with untreated HIV infection and in non-HIV-infected controls. We also assessed phenotypic differences between CD31+ and CD31- subsets in memory and terminally differentiated (TEMRA) CD4+ and CD8 + T cells.Forty-one individuals with untreated HIV infection and 34 non-HIV-infected controls were included in the study. We compared the expression of CD31 in CD4+ and CD8 + T cells across stages of differentiation in the two study groups by flow cytometry. We also analyzed the expression of CD57 (a marker of senescence), Ki67 (a marker of cycling cells), PD-1 (a marker of exhaustion), and CD38/HLA-DR (a marker of immune activation) on memory and TEMRA CD31+ and CD31- T cells.CD31 expression was significantly higher in CD8 + T cells than in CD4 + T cells, measured as frequency, absolute numbers and median fluorescence intensity (MFI), in both study groups (p0.0001 in all cases). Intermediate differentiation subsets of CD4+ and CD8 + T cells expressed higher levels of CD31 in the context of HIV infection (p0.001 in all cases). CD31 expression frequency decreased with cellular differentiation of CD4+ and CD8 + T cells in both groups, but this decrease was steeper in individuals without HIV infection (CD4+: p0.001 and CD8+: p0.0001). As expected, memory and TEMRA CD4+ and CD8 + T cells expressed significantly higher levels of CD57, PD-1, Ki67 and CD38/HLA-DR in HIV-infected compared to non-HIV-infected individuals (p0.01 in all cases). CD31 expression was associated with lower activation of memory (but not TEMRA) CD4 + T cells in non-HIV-infected persons, an effect not observed in the HIV-infected group. CD31 expression on memory CD8 + T cells of HIV-infected individuals was associated higher levels of PD-1 (p = 0.0019) and CD38/HLADR (p = 0.0345), and higher PD-1 expression on CD8 + TEMRA (p = 0.0024), an effect not observed in non-HIV-infected individuals.In the context of HIV-associated chronic immune activation, specifically on memory CD8 + T cells, CD31 expression was associated with higher PD-1 and CD38/HLA-DR co-expression, suggesting that CD31 expression may result from an insufficient attempt to contain T cell exhaustion and activation. CD31-targeted therapies may contribute to modulate these cellular responses.

Impaired wound healing is responsible for significant morbidity and mortality worldwide. It is necessary to find a stable, efficient, and safe method to promote soft tissue wound healing. Fat grafting has become increasingly popular in contouring procedures. However, more recently, there has been an emphasis on its regenerative potential. In this study, we established the wound healing model using nude mice. Hematoxylin and eosin and Masson stainings were performed to assess the effect of chyloid fat on the histology of wound healing. A laser Doppler perfusion imager was used to evaluate the blood perfusion of wounds. Immunohistochemistry was carried out to detect the expression of CD31 in wound tissues. The results suggested that after treatment with granule fat or chyloid fat, wound healing was accelerated and blood perfusion was promoted. In addition, granule fat or chyloid fat treatment promoted the angiogenesis of the wound. In addition, we evaluated the amount of adipose-derived mesenchymal stem cells in chyloid fat and granule fat. It was found that chyloid fat contained more adipose-derived mesenchymal stem cells than granule fat did. In conclusion, we proved that chyloid fat could significantly accelerate the wound healing process via promoting angiogenesis. The adipose-derived mesenchymal stem cell plays a critical role in this effect of chyloid fat.

Background Adipose tissue engineering greatly reduce the burden of reconstruction surgery. Subcutaneous transplantation of decellularized adipose tissue was capable of recellularization. However, further improvements are required to promote angiogenesis and adipogenesis. Thus, the authors proposed a neo-mechanical protocol to isolate adipose tissue-derived extracellular vesicles (ATEVs) from liposuction as a factor for both angiogenesis and adipogenesis, and prepared ATEV-rich decellularized adipose tissue hydrogel for adipose tissue engineering. Methods Adipose liquid extract and lipid-devoid adipose tissue were extracted by means of homogenization and repeated freezing and thawing. ATEVs were isolated from adipose liquid extract by ultracentrifugation. Decellularized adipose tissue hydrogel was prepared by optimized decellular technique. The optimum dose of ATEVs for promoting angiogenesis and adipogenesis was screened out by co-culture with vascular endothelial cells and 3T3-L1 cells, then mixed with the hydrogel at the optimal dose. ATEV-enriched hydrogel was injected subcutaneously into the right back of severe combined immunodeficiency mice, and hydrogel was injected as a control on the contralateral side. ATEVs were supplementarily injected into the newborn tissue on the right back at postoperative day 14, and the same amount of phosphate-buffered saline was injected into the left as a control. The samples of both sides were harvested at postoperative weeks 2, 4, and 8 and subjected to volume measure, hematoxylin and eosin staining, and immunofluorescence (CD31 and perilipin) staining. Results The optimum dose of ATEVs for promoting angiogenesis and adipogenesis was 50 μg/ml. In vivo, supplementing ATEVs increased the volume of decellularized adipose tissue hydrogel specimens compared with control specimens at postoperative week 4 (99.4 ± 22.5 μl versus 126.1 ± 16.7 μl; n = 8; p Conclusion ATEV-enriched adipogenic hydrogel encourages enhanced angiogenesis and adipogenesis and could serve as a promising biomaterial for adipose tissue engineering.

Vasonatrin peptide (VNP) is a synthetic peptide that possesses vasodilatory, natriuretic, and anti-inflammatory properties. The authors aimed to analyze the effects of VNP on fat graft survival. Twenty Sprague–Dawley rats are randomly divided into two groups of 10. Fat grafts are harvested from the right inguinal region. After preparation, fat grafts are placed to the interscapular region. The first group of rats were administered VNP after their fat injection, while the second group received tail-vein injections of an equal volume of sterile saline following their fat injection. Experiment and control groups are evaluated according to their level of degeneration of adipocytes, fat necrosis, vacuolization, cyst formation in adipocytes, fibrosis of the fat tissue, capillary density, and CD31 immunohistochemical staining. Degeneration, vacuolization, and cyst formation in adipocytes were lower in the experiment group. Increased capillary density in the experiment group was demonstrated by CD31 antibody staining and by counting capillary density under a microscope. The average percentage of change in weight of the fat grafts in the experiment group was lower than that in the control group. The results indicate that VNP has some beneficial effects on fat graft survival by multiple independent mechanisms that influence both local and systemic homeostasis.

We present a challenging case of HIV-related lymphangioma-like Kaposi sarcoma (LLKS) affecting the oral cavity. A 54-year-old Brazilian male patient was referred to our center complaining of bleeding lesions affecting the oral cavity for 2 months. Interestingly, these oral lesions were the first clinical manifestation of HIV infection. Clinically, multiple erythematous nodular and patch lesions were observed. An incisional biopsy was performed, revealing numerous microscopic angled and irregular vascular channels lined with flattened endothelial cells. More cellularized and solid areas consisting of more fusiform cells with little pleomorphism and with slit-like vascular channels were noted. Based on immunoreactivity for CD31, CD34, D2-40, and HHV-8, the final diagnosis was oral LLKS. Highly active antiretroviral therapy (HAART) was initiated with dolutegravir, tenofovir, and lamivudine. During follow-up, the patient showed KS metastases to other sites and a chemotherapeutic protocol was initiated. Regression of the oral lesion was clearly noted by the clinicians 1 year after the KS diagnosis. Dentists should be able to recognize systemic diseases that affect the oral cavity such as KS in order to make an early diagnosis of its oral manifestations and to implement effective therapeutic measures to ensure a better prognosis.

Objective We investigated the role of angiogenesis and vascular permeability in the pathogenesis of human drug-resistant epilepsy due to hippocampal sclerosis. Methods Resected hippocampi from 30 histologically confirmed cases of hippocampal sclerosis and 30 age-matched post-mortem controls were examined by immunohistochemical quantitation of vascular endothelial markers, CD31 and CD105 (markers of newly formed vessels), and data were analysed relative to MR volumetry. The blood-brain barrier was evaluated based on immunohistochemistry for IgG, albumin, VEGF and AQP4. Results Mean vascular density in the hippocampus was 8.71/mm2 in hippocampal sclerosis samples compared to 7.94/mm2 in age-matched controls. No statistically significant increase in vascular density was found in hippocampal sclerosis samples. Although no neoangiogenesis was found in hippocampal sclerosis samples based on CD105, breakdown of the blood-brain barrier, enhanced neuronal expression of VEGF, and perivascular seepage of IgG and albumin with uptake within neurons and astrocytes were found. Redistribution of the water channel protein, AQP4, reflected by change from normal punctate labelling to intense diffuse staining in hippocampal sclerosis samples, indicated an altered glia-vascular interface, disrupting blood-brain barrier permeability. Significance Our data show no objective histological evidence of angiogenesis in hippocampal sclerosis samples. When controlled for the confounding variable of hippocampal area, there was no difference in vascular density between cases and controls. A leaky blood-brain barrier and redistribution of AQP4 were identified which may contribute to epileptogenesis. This constitutes the largest study in the published literature evaluating a role of vascular permeability and angiogenesis in human hippocampal sclerosis.

In recent decades, epidemiological, clinical, and experimental studies have demonstrated that a diet with antioxidant or anti-inflammatory function plays a central role in the prevention of atherosclerosis (AS). The purpose of this study was to explore the effects of Cannabis seed oil (CO) administration on in vitro antioxidant capacity as well as blood lipid profiles, lipid peroxidation, inflammatory response, and endothelial cell integrity. Female ApoE-/- mice were fed a high-cholesterol diet and administrated with CO or phosphate-buffered saline (PBS) and seal oil by gavage for 8 weeks. The results show that CO administration reduced the levels of serum triglycerides and low-density lipoprotein cholesterol at week 6. Additionally, a decrease in serum tumor necrosis factor α and nitric oxide was also observed. Moreover, results from CD31 staining and scanning electron microscopy revealed that CO treatment alleviated the endothelial cell damage and lipid deposition induced by a high-cholesterol diet. The ratio of lesion area to the total aorta area was 19.57% for the CO group, which was lower than the PBS control group (24.67%). Collectively, CO exerted anti-atherosclerotic effects by modulating serum lipid profiles and inflammatory responses and improving endothelial cell integrity and arterial lipid deposition. The results provide a promising preventive strategy for the early progression of AS.

The present study was to investigate whether dihydroartemisinin (DHA), which is a highly effective and safe drug in the treatment of malaria, could be repurposed for the treatment of skin fibrosis and vascular dysfunction in systemic sclerosis (SSc).The value of DHA was determined using a bleomycin-induced model of skin fibrosis. mRNA transcriptome analysis was performed, and the targets of DHA on fibroblasts were identified. Immunofluorescence staining was used to identify dermal vessels undergoing endothelial-to-mesenchymal transition (EndoMT). Autophagic flux was detected by western blot and mRFP-GFP-LC3 adenovirus vector transfection.Both systemic and topical administration of DHA decreased dermal thickness and collagen deposition and alleviated EndoMT in bleomycin-induced skin fibrosis mice model. Treatment of human umbilical vein endothelial cells (HUVECs) with TGF-β1 resulted in the acquisition of the activation marker (α-SMA) and loss of endothelial markers (CD31 and VE-cadherin), a process that was restored by DHA. DHA significantly suppressed skin fibroblast activation and collagen-1 production mainly through regulating PI3K-Akt pathway. DHA also induced fibroblast autophagic flux and that autophagy dependently suppressed collagen-1 production.The results of the present study revealed that oral and topical DHA administration ameliorated tissue fibrosis and protected dermal blood vessels from bleomycin-induced EndoMT. Our study has elucidated the value of repurposing DHA for the treatment of SSc. Key Points • Oral or topical usage of DHA alleviated dermal fibrosis and EndoMT in bleomycin-induced skin fibrosis mice models. • DHA autophagy dependently inhibited fibroblast activation and collagen deposition via PI3K-ATK pathway. • DHA inhibited EndoMT of HUVECs induced by TGF-β1 by the downregulation of α-SMA and the upregulation of CD31 and VE-cadherin.

Background In vivo prevascularization followed by pedicled transfer has emerged as a promising strategy for tissue engineering in recent years. We recently demonstrated that capsule tissue could serve as a novel axial in vivo vascular bed, although its high-density microvessels could only be maintained for about a week. Objectives In this present study, we aimed to demonstrate whether low-intensity pulsed ultrasound (LIPUS) promotes angiogenesis in capsule tissue. Material and methods After successful induction of capsule tissue using a skin expander, 24 rabbits were randomly divided into the LIPUS group and the control group. The LIPUS group received LIPUS treatment 3 times per week. After 2 and 4 weeks of treatment, angiogenesis of the capsule tissue was assessed using in vivo and in vitro methods, including contrast-enhanced ultrasound (CEUS), photoacoustic imaging (PAI), photoacoustic microscope (PAM), and CD31 immunohistochemistry. Results In vivo assessments (CEUS, PAI and PAM) showed that tissue perfusion, hemoglobin content and vascular density were all significantly higher in the LIPUS group, which was consistent with CD31 immunohistochemistry. The LIPUS also promoted protein and mRNA expression of vascular endothelial growth factor α (VEGFα) and basic fibroblast growth factor (bFGF) in capsule tissue. Furthermore, cell experiments showed that LIPUS enhanced tube formation of human microvascular endothelial cells (HMECs) and promoted secretion of VEGFα and bFGF. Conclusions The LIPUS treatment promoted angiogenesis of the capsule tissue by stimulating release of angiogenic factors such as VEGFα and bFGF from endothelial cells, making the capsule tissue more potent and sustained when acting as in vivo vascular bed.

The retinal vascular endothelial cells can be damaged by oxidative stress even in the early stage of diabetic retinopathy (DR). This study aimed to investigate the protective effect of curcumin on the rat retinal vascular endothelial cells (RRVECs) in high glucose circumstance. The cultured RRVECs were identified and characterized by both of vWF and CD31 immunofluorescence expression. The activation of ROS/NF-κB signal pathway was examined by electrophoretic Mobility Shift Assay (EMSA), immunohistochemistry and Western blot; the apoptosis of RRVECs was tested by flow cytometry. We found that curcumin reduced the reactive oxygen species (ROS) and relieved the apoptosis in RRVECs exposed to the high glucose by flow cytometry. It was revealed that the increased activity of NF-κB and phosphorylated NF-κB in RRVECs induced by high glucose concentration was significantly suppressed by curcumin. We concluded that curcumin could suppress the oxidative stress via regulation of NF-κB signal to protect the RRVECs in DR.

The present study was undertaken to investigate whether Cu protects vasculatures from ischemic injury in the heart. C57/B6 mice were introduced to myocardial ischemia (MI) by permanent ligation of the left anterior descending (LAD) coronary artery. Two hours post-LAD ligation, mice were intravenously injected with a Cu-albumin (Cu-alb) solution, or saline as control. At 1, 4, or 7 days post-MI, hearts were collected for further analysis. A dramatic decrease in CD31-positive endothelial cells concomitantly with abundant apoptosis, along with obstruction of blood flow, was observed in ischemic myocardium 1 day post-MI. The early Cu-alb treatment protected CD31-positive cells from apoptosis, along with a preservation of micro-vessels and a decrease in infarct size. This early vasculature preservation ensured myocardial blood perfusion and protected cardiac contractile function until 28 days post-MI. This strategy of Cu-alb treatment immediately following MI would help develop a therapeutic approach for acute heart attack patients in a clinical setting.

Objective To demonstrate the existence of lymphatics in the canine anterior uvea using lymphatic-specific markers Lyve-1, Prox-1, and podoplanin, the endothelial cell marker CD31, and basement membrane matrix marker collagen IV. Design Prospective Study. Animals Eight normal globes from animals euthanized for unrelated health problems. Procedures Sagittally cut serial sections of six normal canine eyes were immunofluorescence double-stained with Lyve-1 and CD31 and single-stained with colorimetric Prox-1 and collagen IV. Three serial sections from 2 additional eyes were cut in the coronal plane at the level of the ciliary body and immunofluorescence double-stained with Lyve-1 and CD31 to map lymphatic channel distribution. Lymphatics from normal canine lymph nodes were used for validation of podoplanin. Results Four of 6 of the sagitally sectioned eyes had Lyve-1-positive lymphatic-like structures that were distinct from CD31-positive blood vessels in the iris base and ciliary body. Both of the coronally sectioned globes had Lyve-1-positive lymphatic-like structures in the ciliary body. The location of these structures was evaluated and found to be diffusely present circumferentially around the ciliary body. Conclusion and clinical relevance These results support the existence of lymphatic channels in the anterior uveal tract of the canine eye. This could indicate the presence of a novel uveolymphatic outflow pathway, which may play a role in aqueous humor outflow. Future studies are needed to confirm the existence and elucidate the role of this proposed uveolymphatic outflow pathway and potentially develop novel treatment options for managing glaucoma.

This study tested the hypothesis that combined therapy with human umbilical cord‐derived mesenchymal stem cells (HUCDMSCs) and hyperbaric oxygen (HBO) was superior to either one on preserving neurological function and reducing brain haemorrhagic volume (BHV) in rat after acute intracerebral haemorrhage (ICH) induced by intracranial injection of collagenase. Adult male SD rats (n = 30) were equally divided into group 1 (sham‐operated control), group 2 (ICH), group 3 (ICH +HUCDMSCs/1.2 × 106 cells/intravenous injection at 3h and days 1 and 2 after ICH), group 4 (ICH +HBO/at 3 hours and days 1 and 2 after ICH) and group 5 (ICH +HUCDMSCs‐HBO), and killed by day 28 after ICH. By day 1, the neurological function was significantly impaired in groups 2‐5 than in group 1 (P

Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

Background Myocardial ischemia-reperfusion (I/R) injury is still thought to be an unsolved puzzle that may lead to reperfusion arrhythmias and sudden cardiac death. Inflammation plays a key role in myocardial I/R. Studies have indicated that purinoceptor 2Y12 (P2Y12) antagonists have anti-inflammatory properties that are cardioprotective. Objectives In this study, we explored whether inhibition of P2Y12 in macrophages could reduce cardiac inflammation and attenuate reperfusion arrhythmias after myocardial I/R. Material and methods Rats were randomly divided into 4 groups: group A (control + vehicle); group B (control + P2Y12 shRNA lentiviral vector); group C (myocardial I/R + vehicle); and group D (myocardial I/R + P2Y12 shRNA lentiviral vector). Infarct size, reperfusion arrhythmias, and P2Y12 and platelet endothelial cell adhesion molecule-1 (CD31) protein expression were measured. Results The incidence of reperfusion ventricular tachycardia and fibrillation (VT/VF) was 90% in the I/R group, while it was reduced to 50% by P2Y12 shRNA treatment. Ionized calcium binding adapter molecule 1 and P2Y12 immunoreactivity in the myocardial I/R + P2Y12 shRNA group was lower compared to the myocardial I/R group. P2Y12 shRNA treatment increased α-smooth muscle actin (α-SMA) and CD31 protein expression, as evidence by western blot and immunohistochemistry analyses (0.31 ±0.01 compared to 0.26 ±0.008, group D compared to group C, p Conclusions Inhibition of P2Y12 in macrophages improved reperfusion arrhythmias in our rat I/R model, suggesting that blocking P2Y12 could decrease the inflammatory response after cardiac perfusion.

We examined the effect of microRNA-320b (miR-320b) on tumor growth and angiogenesis in lung cancer and also determined its downstream molecular mechanisms. Lung cancer tissues and adjacent non-cancerous tissues were collected from 66 patients with lung cancer. miR-320b expression was experimentally determined to be expressed at low level in cancer tissues. The results of gain-of-function experiments suggested that miR-320b overexpression suppressed cancer cell invasion, tube formation, tumor volume and angiogenesis in xenografted nude mice. Hepatocyte nuclear factor 4 gamma (HNF4G) was identified as a target of miR-320b based on in silico analysis. Dual-luciferase reporter gene assays further identified the binding relationship between HNF4G and miR-320b. Lung cancer tissues exhibited increased expression of HNF4G and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Meanwhile, HNF4G knockdown suppressed IGF2BP2 expression, thereby repressing cancer cell invasion and tube formation. Furthermore, IGF2BP2 modified m6A to increase the expression of thymidine kinase 1 (TK1), thus promoting angiogenesis. In nude mice, restoration of TK1 reversed the suppressive effect of miR-320b overexpression on tumor growth rate and CD31 expression. In conclusion, miR-320b suppresses lung cancer growth and angiogenesis by inhibiting HNF4G, IGF2BP2 and TK1.

Introduction: Tumor agiogenesis is generally classified in to two types, namely, sprouting and intussusceptive angiogenesis. The formation of new blood vessels from the already existing is termed as sprouting angiogenesis. Many growth factors including endothelial growth factor (VEGF) contribute the formation of new vessels at the tumor sites. On other hand, the tumor able to split the existing blood vessels, this type is termed as intussuscepted angiogenesis and discovered in human colon adenocarcinoma xenograft. In intussuscepted angiogenesis, an existing blood vessel splits into two new blood vessels by formation of trans vascular pillars. Objective: The present study aimed to analyse the nicrovascular density on breast cancer patients using CD 34 immunostaining method. Results: This mis-regulation may lead to the development of cancers. Histological grading with Grade II were more with 19 (63.3%) cases followed by Grade I with 4 (13.3%) and Grade III with 3 (10%) cases. There was increase in mean vascular density in Grade II when compared with Grade I and Grade III. However no significant correlation was observed statistically with a P value of 0.176. Using different antibodies such as CD34, CD31, Factor VIII and CD105 to microvessels differentiation was highlighted. Conclusion: The results showed that the anti-CD34 monoclonal antibody is more sensitive than the anti- CD31 antibody in calculation of MVD in breast cancer as mentioned in previous studies.

Research on chronic ischemic stroke is limited. One of the more promising approaches showing positive effects in the acute stage is mononuclear bone marrow cell therapy. This research may be the first which presents data about the optimum dose of bone marrow mononuclear cells (BM-MNCs) for chronic ischemic stroke in rats and discusses factors influencing recovery in the chronic stage. We performed temporary middle cerebral artery occlusion (MCAO) procedures on the rats which were then randomly assigned to one of two experimental groups in which they were given either low or high doses of autologous BM-MNCs (5 million or 10 million cells per kg body weight). Rat brains were fixed for HE, CD31, and doublecortin staining for analysis of the effects. Rat behavior was assessed weekly using the cylinder test and a modified neurological severity score (NSS) test. In the four weeks prior to administration of BM-MNC, cylinder test scores improved to near normal, and NSS test scores improved moderately. The infarct zone decreased significantly (p

Purpose. Postischemic inflammation induces angiogenesis, while platelet P2Y12 inhibitors can alleviate this inflammation. Therefore, we studied the potential effects of P2Y12 inhibitor, ticagrelor, on angiogenesis in a mouse model of hindlimb ischemia. Methods. Laser Doppler perfusion imaging and capillary density measurement were used for angiogenesis quantified. Immunofluorescence was used to detect the level of CD31. The mice muscle was harvested for enzyme-linked immunosorbent (ELISA) assay of interleukin- (IL) 10 activity and Western blot determination of vascular endothelial growth factor (VEGF) production. Results. Ischemic hindlimb angiogenesis was sharply decreased in IL-10+/+ mice than IL-10-/- mice. Ticagrelor inhibited angiogenesis and blood reperfusion recovery significantly elevated the levels of IL-10 and decreased the expression of VEGF in the IL-10+/+ mouse ischemic hindlimb, which were abolished in IL-10-deficient (IL-10–/–) C57BL/6J mice. Conclusion. The study underscores that the effect of ticagrelor antiangiogenic function is related with the higher IL-10 expression.

Oral tongue squamous cell carcinoma (OTSCC) is one of the most common cancers worldwide and is characterized by early metastasis and poor prognosis. Recently, we reported that extracellular interleukin‐17F (IL‐17F) correlates with better disease‐specific survival in OTSCC patients and has promising anticancer effects in vitro. Vasculogenic mimicry (VM) is the formation of an alternative vasculogenic system by aggressive tumor cells, which is implicated in treatment failure and poor survival of cancer patients. We sought to confirm the formation of VM in OTSCC and to investigate the effect of IL‐17F on VM formation. Here, we showed that highly invasive OTSCC cells (HSC‐3 and SAS) form tube‐like VM on Matrigel similar to those formed by human umbilical vein endothelial cells. Interestingly, the less invasive cells (SCC‐25) did not form any VM structures. Droplet‐digital PCR, FACS, and immunofluorescence staining revealed the presence of CD31 mRNA and protein in OTSCC cells. Additionally, in a mouse orthotopic model, HSC‐3 cells expressed VE‐cadherin (CD144) but lacked Von Willebrand Factor. We identified different patterns of VM structures in patient samples and in an orthotopic OTSCC mouse model. Similar to the effect produced by the antiangiogenic drug sorafenib, IL‐17F inhibited the formation of VM structures in vitro by HSC‐3 and reduced almost all VM‐related parameters. In conclusion, our findings indicate the presence of VM in OTSCC and the antitumorigenic effect of IL‐17F through its effect on the VM. Therefore, targeting IL‐17F or its regulatory pathways may lead to promising therapeutic strategies in patients with OTSCC., In this study, we first confirmed the presence of the vasculogenic mimicry (VM) phenomenon in oral cancer patients, in vitro and in vivo, which exhibited different molecular patterns and markers. Next, we reported a novel therapeutic effect of IL‐17F against VM formation in highly aggressive cancer cells. Overall, targeting IL‐17F or its regulatory pathways could lead to promising therapeutic strategies in patients with oral cancer and other solid tumors as well.

Introduction Stem cell transplantation is one of the most promising strategies to improve healing in chronic wounds as systemic administration of endothelial progenitor cells (EPC) enhances healing by promoting neovascularization and homing though a high amount of cells is needed. In the following study, we analysed whether local application can reduce the number of EPC needed achieving the same beneficial effect on wound healing. Material and Methods Wound healing after local or systemic treatment with EPC was monitored in vivo by creating standardized wounds on the dorsum of hairless mice measuring wound closure every second day. Systemic group received 2 × 106 EPC i.v. and locally treated group 2 × 105 EPC, locally injected. As control PBS injection was performed the same way. Expression of CD31, VEGF, CD90 and, SDF-1α was analysed immunohistochemically for evaluation of neovascularisation and amelioration of homing. Results Local (7.1 ± 0.45 SD) as well as systemic (6.1 ± 0.23 SD) EPC transplantation led to a significant acceleration of wound closure compared to controls (PBS local: 9.7 ± 0.5 SD, PBS systemic 10.9 ± 0.38 SD). Systemic application enhanced CD31 expression on day 6 after wounding and local EPC on 6 and 9 in comparison to control. VEGF expression was not significantly affected. Systemic and local EPC treatment resulted in a significantly enhanced SDF-1α and CD90 expression on all days investigated. Conclusion Local as well as systemic EPC treatment enhances wound healing. Moreover, beneficial effects are obtained with a tenfold decrease number of EPC when applied locally. Thus, local EPC treatment might be more convenient way to enhance wound healing as number of progenitor cells is limited.