Vascular Endothelial Cell Marker Research Articles

Maternal supplementation spermidine during gestation improves placental angiogenesis and reproductive performance of high prolific sows

Spermidine (SPD) is a widely recognized polyamine compound found in mammalian cells and plays a key role in various cellular processes. We propose that SPD may enhance placental vascular development in pregnant sows, leading to increased birth weight of piglets. Six hundred and nine sows at 60 days of gestation were randomly assigned into a basal diet (CON group), basal diet supplemented 10 mg/kg of SPD (SPD1 group), and basal diet supplemented 20 mg/kg of SPD (SPD2 group), respectively. Compared with the CON, SPD1 significantly increased the average number of healthy piglets per litter and the placental efficiency (P < .05), while the average number of mummified fetus per litter and the percentage of weak piglets significantly decreased (P < .05). In the plasma metabolomics, SPD content in plasma of sows (P = .075) and umbilical cord plasma of piglets (P = .078) had an increasing trend in response to SPD1. Furthermore, SPD1 increased the expression of the vascular endothelial cell marker protein, platelet endothelial cell adhesionmolecule-1 (PECAM-1/CD31) and the density of placental stromal vessels (P < .05). Moreover, as compared to CON, SPD2 significantly decreased the average number of mummified fetus per litter (P < .05), while the placental efficiency and the expression of amino acid transporter solute carrier (SLC) family 7, member7 (SLC7A7) and glucose transporters SLC2A2) and SLC5A4 in placental tissue significantly increased (P < .05). These results suggest that maternal supplementation of SPD during pregnancy increased healthy litter number, and promoted placental tissue development. Our findings provide evidence that maternal SPD has the potential to improve the production performance of sows.

Repeated treatment with VEGF receptor inhibitors induces phenotypic changes in endothelial cells and pericytes in the rat retina

Abnormal ocular angiogenesis is a major cause of visual impairment and vision loss in neovascularization-related diseases. Currently, anti-vascular endothelial growth factor (VEGF) drugs are used to treat ocular neovascularization, but repeated injections are needed to maintain their therapeutic effects. However, repeated injection of anti-VEGF drugs may affect the retinal blood vessel phenotype and diminish therapeutic effects. In this study, we aimed to investigate the phenotypic changes in endothelial cells and pericytes caused by the repeated interruption of the VEGF receptor signaling pathway in neonatal rats. KRN633 (10 mg/kg), a VEGF receptor tyrosine kinase inhibitor, was subcutaneously administered on postnatal day (P)-7 and P8 (first round), P14 and P15 (second round), and P21 and P22 (third round). The rat eyes were collected on P7, P9, P14, P16, P21, P23, P28, and P35. Using retinal flat-mount specimens stained with specific markers for vascular endothelial cells, basement membranes, and pericytes, the arteriolar tortuosity, capillary area density, and distribution of pericytes were evaluated. Significant loss of capillaries was observed the day after the first round of KRN633 treatment, after which aggressive angiogenesis occurred, leading to the formation of tortuous arterioles. Rats that completed second and third rounds of KRN633 treatment showed more severe abnormalities in the retinal vasculature than those that only completed first round treatment. Repeated treatment with KRN633 decreased the anti-angiogenic effects but increased the immunoreactivity of α-smooth muscle actin in the pericytes on veins and capillaries. α-Smooth muscle actin expression was inversely correlated to anti-angiogenic effects. Overall, these results revealed that repeated interruption of VEGF receptor signaling pathway altered the phenotypes of endothelial cells and pericytes and induced anti-VEGF drug resistance. Therefore, careful follow-up is necessary when using anti-VEGF drugs to treat abnormal angiogenesis-associated ocular diseases.

Human Placenta-Derived Mesenchymal Stem Cells Combined With Artificial Dermal Scaffold Enhance Wound Healing in a Tendon-Exposed Wound of a Rabbit Model.

To overcome the difficulty of vascular regeneration in exposed tendon wounds, we combined human placenta-derived mesenchymal stem cells (hPMSCs) with an artificial dermal scaffold and assessed their role in promoting vascular regeneration and wound healing in vivo. hPMSCs were isolated from the human placenta and characterized based on their morphology, phenotypic profiles, and pluripotency. New Zealand rabbits were used to establish an exposed tendon wound model, and hPMSCs and artificial dermal scaffolds were transplanted into the wounds. The results of gross wound observations and pathological sections showed that hPMSCs combined with artificial dermal scaffold transplantation increased the vascularization area of the wound, promoted wound healing, and increased the survival rate of autologous skin transplantation. Following artificial dermal scaffold transplantation, hPMSCs accelerated the vascularization of the dermal scaffold, and the number of fibroblasts, collagen fibers, and neovascularization in the dermal scaffold after 1 week were much higher than those in the control group. Immunohistochemical staining further confirmed that the expression of the vascular endothelial cell marker, CD31, was significantly higher in the combined transplantation group than in the dermal scaffold transplantation group. Our findings demonstrated that hPMSCs seeded onto artificial dermal scaffold could facilitate vascularization of the dermal scaffold and improve tendon-exposed wound healing.

A novel approach for transforming breast cancer stem cells into endothelial cells.

Tumor vascular endothelial cells play a pivotal in the tumor microenvironment, influencing the proliferation, invasion, and metastasis of tumor progression. The present study investigated a novel method for inducing the transformation of breast cancer stem cells into endothelial cells, providing a cellular model investigating anti-angiogenic mechanisms in vitro. The breast cancer cell line MCF-7 was used, and the expression of CD133 was initially detected using flow cytometry. CD133+ breast cancer cells were purified using immunomagnetic bead sorting technology, yielding an MCF-7CD133+ subpopulation. The proliferation ability of these cells was assessed using an MTT assay, while their microsphere formation ability was evaluated using a microsphere formation assay. Post-transformation in an optimized endothelial cell culture medium, expression of endothelial cell markers CD31 and CD105 were detected using flow cytometry. Endothelial cell tube formation assays and DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) assays were employed to analyze the endothelial cell function of the MCF-7CD133+ cells. MDM2/CEN12 gene amplification was detected through fluorescence in situ hybridization (FISH). The MCF-7 breast cancer cell line exhibited 1.7±0.3% trace cells expressing the stem cell surface marker CD133. After anti-CD133 immunomagnetic bead sorting, MCF-7CD133+ and MCF-7CD133- subpopulation cells were obtained, with CD133 expression rates of 85.6±2.8 and 0.18±0.08%, respectively. MTT assay results demonstrated that, after 7 days, the proliferation rate of MCF-7CD133+ cells was significantly higher compared with MCF-7CD133- cells. MCF-7CD133+ subpopulation cells displayed strong stem cell characteristics, growing in suspension in serum-free media and forming tumor cell spheres. In contrast, MCF-7CD133- cells failed to form microspheres. After culturing cells in endothelial cell differentiation and maintenance media, the percentage of MCF-7CD133+ cells before and after endothelial cell culture was 0.3±0.16 and 81.4±8.37% for CD31+ cells and 0.2±0.08 and 83.8±7.24% for CD105+ cells, respectively. Vascular-like structure formation and Ac-LDL phagocytosis with red fluorescence in the tube formation assays confirmed endothelial cell function in the MCF-7CD133+ cells. FISH was used to verify MDM2/CEN12 gene amplification in the induced MCF-7CD133+ cells, indicating tumor cell characteristics. The modified endothelial cell transformation medium effectively induced differentiated tumor stem cells to express vascular endothelial cell markers and exhibit endothelial functions, ideal for in vitro anti-angiogenesis research.

Lymphatic vessels accompanying dorsal and basal dural sinuses in the human brain

Recent investigations showed the presence of meningeal lymphatic vessels (mLVs) along the superior sagittal and transverse dural sinuses which drain both fluid and immune cells from the cerebrospinal fluid (CSF) to the deep cervical lymph nodes. This study uses immunohistochemistry (IHC) and the Western Blot technique to show the presence of mLV accompanying the dorsal (superior sagittal, inferior sagittal, transverse, sigmoid, and straight) and basal (cavernous, sphenoparietal, superior, and inferior petrosal) dural sinuses in the human brain. Samples for IHC were obtained from dorsal and basal meningeal dural sinuses of 3 human cadavers and 3 autopsies. Routine histological techniques were carried out for the specimens. Podoplanin (PDPN, lymphatic vessel endothelial cell marker) and CD31 (vascular endothelial cell marker) IHC staining were applied to the 5 µm thick paraffin sections. Furthermore, PDPN and CD31 protein expressions were evaluated using Western Blot to the tissue samples from the same regions of 4 autopsies. Two consecutive sections from each sinus were PDPN, and CD31 was stained to differentiate blood vessels (BV) from mLV. The IHC staining showed the presence of mLVs accompanying both dorsal and basal dural sinuses. The mLVs accompanying the dorsal dural sinuses had a larger dimensions range compared to the basal dural sinuses. However, the number of mLVs along the basal dural sinuses was more than the mLVs along the dorsal ones. Further, fluid channels were closely localized to the mLV, with varying diameters and densities. Western Blotting technique showed the presence of PDPN expression in both dorsal and basal dural sinus samples. The knowledge of the presence of mLV along both dorsal and basal dural sinuses in humans can increase the understanding of how mLV contributes to the brain lymphatic circulation and may help understand the neuropathophysiological processes of various neurological diseases.

A Novel tRNA-Derived Fragment, tRFGlnCTG, Regulates Angiogenesis by Targeting Antxr1 mRNA.

As a novel non-coding RNA with important functions corresponding to various cellular stresses, the function of tRFs in angiogenesis remains unclear. Firstly, small RNA sequencing was performed on normal and post-muscle injury mouse tibialis anterior muscle to identify and analyse differentially expressed tRF/tiRNA. tRNA GlnCTG-derived fragments (tRFGlnCTG) were found to be overexpressed in high abundance in the damaged muscle. Subsequent in vitro experiments revealed that the overexpression of tRFGlnCTG suppressed the vascular endothelial cells' viability, cell cycle G1/S transition, proliferation, migration, and tube-formation capacity. Similarly, in vivo experiments showed that the tRFGlnCTG decreased the relative mRNA levels of vascular endothelial cell markers and pro-angiogenic factors and reduced the proportion of CD31-positive cells. Finally, luciferase activity analysis confirmed that the tRFGlnCTG directly targeted the 3'UTR of Antxr1, leading to a significant reduction in the mRNA expression of the target gene. These results suggest that tRFGlnCTG is a key regulator of vascular endothelial cell function. The results provide a new idea for further exploration of the molecular mechanisms that regulate angiogenesis.

Hyperbaric oxygen therapy efficacy on mandibular defect regeneration in rats with diabetes mellitus: an animal study

BackgroundThis study aimed to investigate the influence of hyperbaric oxygen therapy on mandibular critical-sized defect regeneration in rats with experimentally induced type I diabetes mellitus. Restoration of large osseous defects in an impaired osteogenic condition such as diabetes mellitus is a challenging task in clinical practice. Therefore, investigating adjunctive therapies to accelerate the regeneration of such defects is crucial.Materials and methodsSixteen albino rats were divided into two groups (n = 8/group). To induce diabetes mellitus, a single streptozotocin dosage was injected. Critical-sized defects were created in the right posterior mandibles and filled with beta-tricalcium phosphate graft. The study group was subjected to 90-min sessions of hyperbaric oxygen at 2.4 ATA, for 5 consecutive days per week. Euthanasia was carried out after 3 weeks of therapy. Bone regeneration was examined histologically and histomorphometrically. Angiogenesis was assessed by immunohistochemistry against vascular endothelial progenitor cell marker (CD34) and the microvessel density was calculated.ResultsExposure of diabetic animals to hyperbaric oxygen resulted in superior bone regeneration and increased endothelial cell proliferation, which were revealed histologically and immunohistochemically, respectively. These results were confirmed by histomorphometric analysis which disclosed a higher percentage of new bone surface area and microvessel density in the study group.ConclusionsHyperbaric oxygen has a beneficial effect on bone regenerative capacity, qualitatively and quantitively, as well as the ability to stimulate angiogenesis.

Continuous Hypobaric Hypoxia may Promote Atherosclerosis Progression in Apolipoprotein E-deficient Mice.

Background: Intermittent normobaric hypoxia can promote the progression of atherosclerotic plaques. However, the effect of continuous hypobaric hypoxia (CHH), which is a major feature of high-altitude environment, on atherosclerosis has not been investigated thoroughly. Materials and Methods: After eight weeks of high-cholesterol diet, 30 male ApoE-/- mice were randomly divided into control and CHH groups. Mice in the CHH group lived in a hypobaric chamber with an oxygen content of 10% and air pressure of 364 mmhg (equal to 5,800 m altitude above sea level) for 4 weeks, while mice in the control group lived in normoxia condition. Then all mice were euthanized and the atherosclerotic lesion size and plaque stability in the aortic root were assessed. Intraplaque angiogenesis was characterized by immunostaining of CD31 and endomucin, which are identified as specific markers of vascular endothelial cells. Immunohistochemistry and qRT-PCR were performed to measure inflammatory cytokines. Results: Four weeks of CHH exposure promoted the growth of atherosclerotic lesions (p=0.0017) and decreased the stability of atherosclerotic plaques. In CHH group, plaque smooth muscle cells and collagen contents decreased, while plaque macrophages and lipids contents increased significantly (p<0.001). The contents of CD31 (p=0.0379) and endomucin (p=0.0196) in the plaque was higher in the CHH group and correlated with angiogenesis progression. Further, the content of monocyte chemotactic protein-1 (p=0.0376) and matrix metalloproteinase-2 was significantly higher (p=0.0212) in the CHH group. Conclusions: CHH may accelerate atherosclerosis progression in ApoE-/- mice by promoting angiogenesis and inflammation.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats.

To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Dynamic changes of inducible nitric oxide synthase expression in rat’s retina and its role on blood-retinal barrier injury after acute high intraocular pressure

To clarify the role of inducible nitric oxide synthase (iNOS) in blood-retinal barrier (BRB) injury after acute high intraocular pressure (IOP) in rats. Forty-two Sprague-Dawley (SD) rats were randomized into 7 groups [control (Cont), 3, 6, 12, 24, 48, and 72h, n=6]. Except Cont group, other groups' retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of iNOS and tight junction protein zonula occludens (ZO)-1 was detected by Western blotting. Evans blue (EB; 3% ) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6h, 12h groups, the expression of iNOS was localized by immunofluorescence. In order to verify the role of iNOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor (Inh), 6h and 6h+Inh, n=9]. After treatment with the iNOS-specific inhibitor 1400W, the expression of iNOS and ZO-1 and the leakage of BRB were detected again. The immunofluorescence results showed that the expression of iNOS was observed in the Cont group and 6h group, but not in the 12h group. iNOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker NeuN but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period (3h, 6h) after acute high IOP, the expression of iNOS was upregulated, then the down-regulation of iNOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous down-regulation after 6h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3h after acute high IOP. At 6h, the leakage of EB was lower, but at 12h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of iNOS was down-regulated after 1400W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6h group, and significantly down-regulated in the 6h+Inh group, and the leakage of EB was significantly increased after 1400W treatment. These results suggest that the upregulation of iNOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Aberrant Expression of ACO1 in Vasculatures Parallels Progression of Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by mitochondrial dysfunction. However, details about the non-mitochondrial enzymes that sustain the proliferative nature of IPF are unclear. Aconitases are a family of enzymes that sustain metabolism inside and outside mitochondria. It is hypothesized that aconitase 1 (ACO1) plays an important role in the pathogenesis of IPF given that ACO1 represents an important metabolic hub in the cytoplasm. Objectives: To determine if ACO1 expression in IPF lungs shows specific patterns that may be important in the pathogenesis of IPF. To determine the similarities and differences in ACO1 expression in IPF, bleomycin-treated, and aging lungs. Methods: ACO1 expression in IPF lungs were characterized and compared to non-IPF controls by western blotting, immunostaining, and enzymatic activity assay. ACO1-expressing cell types were identified by multicolor immunostaining. Using similar methods, the expression profiles of ACO1 in IPF lungs versus bleomycin-treated and aged mice were investigated. Measurements and main results: Lower lobes of IPF lungs, unlike non-IPF controls, exhibit significantly high levels of ACO1. Most of the signals colocalize with von Willebrand factor (vWF), a lineage marker for vascular endothelial cells. Bleomycin-treated lungs also show high ACO1 expressions. However, most of the signals colocalize with E-cadherin and/or prosurfactant protein C, representative epithelial cell markers, in remodeled areas. Conclusions: A characteristic ACO1 expression profile observed in IPF vasculatures may be a promising diagnostic target. It also may give clues as to how de novo angiogenesis contributes to the irreversible nature of IPF.

13-OR: Cystic Fibrosis Donors Exhibit Striking Decrease in Pancreatic Islet and Exocrine Capillaries

Glucose intolerance and insulin deficiency are common features of cystic fibrosis (CF) , with nearly half of adult CF patients developing CF-related diabetes (CFRD) , but the underlying pathophysiology remains poorly understood. Extensive destruction of the exocrine pancreas occurs in most CF patients. However, islets are still readily detectable, suggesting β cell loss is not the major driver of impaired insulin release. Islets are normally highly vascularized, and islet capillaries are the source of key growth factors that maintain β cell health and function. Islet capillary morphology is altered in type 1 and type 2 diabetes, with increased capillary density and/or vessel thickening being observed. In the present study we aimed to determine whether islet capillaries are also disrupted in CF/CFRD. Pancreas sections (8 control and 8 CF/CFRD, age-matched) were stained for CD31, a marker of vascular endothelial cells. Islets in microscopy images were manually identified (17.8±2.8[SEM] per sample) and CD31-positive area within islet and exocrine was quantified. Mean islet cross-sectional area did not significantly differ between CF and non-CF pancreas specimens. CD31 positive area, expressed as a fraction of islet area, was significantly decreased in CF pancreas specimens (2.8±0.9% vs. 6.1±1.2%; p=0.05) . Islet capillary density (the number of CD31+ objects normalized to islet area) was also decreased (0.88±0.17x103/μm2 vs. 3.8±0.57x103/μm2; p=0.0004) , as was exocrine tissue CD31 positive area (0.17±0.03% vs. 1.5±0.23%; p&amp;lt;0.0001) . Our data demonstrate a significant deficit in islet and pancreatic vascularity in CF/CFRD. This contrasts markedly with the increase in islet capillary density seen in both T1D and T2D and underscores the presence of distinct features of islet morphology that characterize CF. Given that islet capillaries support β-cell function and survival, their loss likely contributes to impaired β-cell function. Disclosure J.J.Castillo: None. A.Aplin: None. R.Akter: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. Funding Cystic Fibrosis Foundation Fellowship (CASTIL20F0)

Abstract 12456: Apelin Expressing Endothelial Stem-Like Cells Orchestrate Lung Microvascular Repair in a Model of Lung-Targeted Endothelial Cell Ablation

Introduction: Endothelial damage plays a central role in acute lung injury (ALI) contributing to alveolar leak and inflammatory cell trafficking. In preclinical models of ALI, repair and regeneration of lung vascular endothelium is required for its resolution. Therefore, we sought to define the cellular and molecular mechanisms underlying lung microvascular regeneration in acute lung injury induced by lung endothelial cell (EC) ablation. Methods and Results: Transgenic mice were created expressing endothelial-targeted human diphtheria toxin receptor. Intratracheal instillation of diphtheria toxin (DT) resulted in the ablation of ~70% of lung endothelial cells, producing severe acute lung injury, with complete resolution by 7 days. Changes in global lung cell populations and gene expression profiles were determined using single-cell RNA sequencing of dissociated lung cells (10x Genomics) at baseline (day 0), 3, 5 and 7 days after lung EC ablation. Single cell analysis revealed 8 distinct EC clusters, including type-A capillary ECs which were characterized by the unique expression of apelin at baseline. DT-induced EC ablation resulted in the emergence of novel endothelial stem-like cells within a transient population of ‘general’ capillary ECs, characterized by the de novo expression of apelin at day 3 post injury. As well, this population exhibited unique expression of the vascular endothelial stem cell marker, protein C receptor, together with the progenitor cell marker, CD34. This was followed by the appearance of proliferative ECs at day 5 expressing apelin receptor and the transcription factor, FoxM1, and these cells were responsible for replenishment of all depleted EC populations. Finally, treatment with an apelin receptor antagonist, ML221, prevented lung microvascular recovery and resulted in death at 4-5 days post DT. Conclusion: Targeted EC ablation revealed a remarkable regenerative capacity of the lung microvasculature. Using single cell transcriptomics, we have demonstrated that lung microvascular repair was orchestrated by newly emergent apelin-expressing endothelial stem-like cells.

Retiform Hemangioendothelioma

Histopathology Histopathological examination is required to confirm the diagnosis of retiform hemangioendothelioma (RH) because RH does not have distinctive clinical features. Histopathological examination reveals arborizing blood vessels in the dermis, extending between the collagen bundles in a reticular fashion and flanked by lymphocytes and hyaline sclerosis. The vessels are lined by monomorphic endothelial cells with scanty cytoplasm and prominent protuberance of the nucleus. The cells can protrude to the vascular lumen like a matchstick or hobnail, and nuclear atypia and mitotic figures are generally absent (Fig. 1).1 Some interspersed lymphocytes can also be seen in and around the blood vessels. Immunohistochemical examination of RH shows positive expression of vascular endothelial cell marker CD34 and weak positive expression of CD31 and FVIII-related antigen.2 This low-grade tumor recurs frequently, but the risk of metastasis is very low.Figure 1: Histopathological features of Retiform Hemangioendothelioma. The epidermis is normal. However, arborizing blood vessels are visible in the dermis. The vessels are lined by monomorphic endothelial cells, and interspersed lymphocytes can be seen in and around the blood vessels. (A) Hematoxylin-eosin, ×100. (B) Hematoxylin-eosin, ×200.Clinical features RH is an extremely rare variant of low-grade angiosarcoma that was first described in 1994 as a distinctive form of angiosarcoma.3 It often occurs in young or middle-aged people and has an unclear pathogenesis. Its main clinical feature is an asymptomatic, slowly growing, well-defined solitary nodular or plaque-like lesion on either the extremities or trunk (Fig. 2).2,4Figure 2: Clinical presentation of Retiform Hemangioendothelioma. Asymptomatic, well-defined, solitary nodular lesion on the lower limb. No abnormality is present in the surrounding skin.The differential diagnoses for RH are angiosarcoma and targetoid hemosiderotic hemangioma. The histopathological features of angiosarcoma are very similar to those of RH. However, angiosarcoma often exhibits nuclear atypia and mitotic figures and the absence of a retiform pattern of blood vessels; additionally, it is more invasive than RH and has a higher risk of recurrence and metastasis. In contrast, targetoid hemosiderotic hemangioma is a superficial and limited lesion, and shoe nail-like vascular endothelial cells can only be seen in some areas.3 These lesions can be identified by histopathologic examination. About 60% of patients with RH develop local recurrence with a tendency for local aggressive behavior. Previous reports have described regional lymph gland metastasis in a single patient and local spread to soft tissue in another patient.5 Only one RH-related death has been reported to date. Surgical excision is the most effective treatment for a circumscribed lesion.6

Angiopoietin-2 regulates vessels encapsulated by tumor clusters positive hepatocellular carcinoma nest-type metastasis via integrin α5β1

Objective: To investigate the molecular mechanism of nest metastasis in blood vessels encapsulated by tumor clusters (VETC) positive hepatocellular carcinoma (HCC). Methods: A total of 72 paraffin embedded HCC tissue samples were collected. Immunohistochemistry staining with CD34 (vascular endothelial cell marker protein) was used to observe the morphological manifestations of VETC cancer nests in primary tumors, bile duct cancerous thrombi and portal vein cancerous thrombi, and to study the characteristics of hematogenous metastasis of VETC cancer nests. Bioinformatics was used to predict the key proteins closely related to VETC cancer nest formation. Immunohistochemistry was used to detect the expression of angiogenin-2 (Ang-2), integrin α5, Integrin β1, and cyclooxygenase-2 (COX-2) proteins in HCC. Transwell cell migration assay was used to detect the effect of Ang-2/integrin α5β1 protein on the migration ability of endothelial cells and HCC cells. Western blotting was used to detect the effect of Ang-2/integrin α5β1 protein on the activity of focal adhesion kinase (FAK) protein. Results: Of the collected HCC specimens, 27 cases (27/72) were VETC (+), including 3 cases with biliary duct cancerous thrombus, 5 cases with portal vein cancerous thrombus, and 3 cases with both biliary duct cancerous thrombus and portal vein cancerous thrombus. VETC (+) HCC could metastasize to portal vein, bile duct, and liver in the form of cancer nest, and the nests retain their intact structure. Ang-2, integrin α5 and integrin β1 were overexpressed in tumor cells and endothelial cells of VETC (+) HCC nests, while COX-2 was only overexpressed in tumor cells of VETC (+) HCC nest. Ang-2 could promote the migration of HCC cell [(121±12) vs (186±11), P<0.01] and endothelial cells [(81±7) vs (163±14), P<0.01]. Integrin α5β1 activation antagonist ATN-161 could significantly block the ability of Ang-2 to promote the migration of HCC cells [(185±10) vs (135±9), P<0.05] and endothelial cells [(156±14) vs (103±6), P<0.05]. ATN-161 could significantly block the phosphorylation of FAK in HCC and endothelial cells induced by Ang-2. Conclusions: VETC (+) HCC could metastasize as a whole in a nested form, and possesses a specific regulatory protein. Ang-2/α5β1/FAK might be potential protein targets in the treatment of VETC (+) HCC nest-type metastasis.

The Application of a Bone Marrow Mesenchymal Stem Cell Membrane in the Vascularization of a Decellularized Tracheal Scaffold.

Airway stenosis is a common problem in the neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU). A tissue-engineered trachea is a new therapeutic method and a research hotspot. Successful vascularization is the key to the application of a tissue-engineered trachea. However, successful vascularization studies lack a complete description. In this study, it was assumed that rabbit bone marrow mesenchymal stem cells were obtained and induced by ascorbic acid to detect the tissue structure, ultrastructure, and gene expression of the extracellular matrix. A vascular endothelial cell culture medium was added in vitro to induce the vascularization of the stem cell sheet (SCS), and the immunohistochemistry and gene expression of vascular endothelial cell markers were detected. At the same time, vascular growth-related factors were added and detected during SCS construction. After the SCS and decellularized tracheal (DT) were constructed, a tetrandrine allograft was performed to observe its vascularization potential. We established the architecture and identified rabbit bone marrow mesenchymal stem cell membranes by 14 days of ascorbic acid, studied the role of a vascularized membrane in inducing bone marrow mesenchymal stem cells by in vitro ascorbic acid, and assessed the role of combining the stem cell membranes and noncellular tracheal scaffolds in vivo. Fourteen experiments confirmed that cell membranes promote angiogenesis at gene level. The results of 21-day in vitro experiments showed that the composite tissue-engineered trachea had strong angiogenesis. In vivo experiments show that a composite tissue-engineered trachea has strong potential for angiogenesis. It promotes the understanding of diseases of airway stenosis and tissue-engineered tracheal regeneration in newborns and small infants.

Creation and grading of experimental corneal scars in mice models

PurposeTo develop a stromal wound healing model and a reliable scar classification score system that correlates photographic evaluation with changes in the structure and organization of the extracellular matrix. Materials and methodsWe tested three stromal injury techniques in adult C57BL/6 mice. Technique 1, a lineal partial thickness keratotomy in the horizontal axis. Technique 2, corneal epithelial and stromal debridement using a diamond burr in the horizontal axis, and technique 3, a combination of techniques 1 and 2. To assess intra-observer and inter-observer agreement between two examiners evaluating formed stromal scars, stereo microscopic photographs of anterior segment were scored by two masked examiners at around 1-month. Depending on the severity of opacification and the area of involvement, scars were classified on a scale from 0 to 3 based on a modified Fantes haze scale. Extracellular matrix composition as well as matrix organization, macrophage infiltration and neovascularization were evaluated with immunofluorescence and second harmonic generation (SHG) microscopy. ResultsTechnique 1 created mild scars, with a score of 0.5 ± 0.43, while techniques 2 (score 2.1 ± 0.45) and 3 (score 2 ± 0.66), created dense scars with a higher score. A significant difference in scar severity score was noted between the 3 techniques (one way ANOVA, p < 0.0001). Masked graders demonstrated excellent agreement (intraclass correlation = 0.927 [95% confidence interval: 0.87–0.96]). The severity of scars noted at stereo microscopy correlated with the severity of changes in extracellular matrix in the stroma as demonstrated by the expression of collagens I, IV and fibronectin and evaluation of matrix hierarchical organization. In contrast to mild scarring, moderate and severe scars had increased expression of CD31 and CD68, markers of vascular endothelial cells and macrophages, respectively. ConclusionMouse models of stromal scarring using simple surgical techniques are described. Corneal scars can be consistently classified by two observers. Grading of scar severity positively correlates with changes in extracellular matrix composition, disorganization and cell infiltration.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Alleviate Lung Injury in Rat Model of Bronchopulmonary Dysplasia by Affecting Cell Survival and Angiogenesis.

Bronchopulmonary dysplasia (BPD) is a serious chronic lung disease in premature newborns, with high morbidity and mortality rates. Mesenchymal stem cell (MSC) transplantation has developed into a promising approach to alleviate BPD. Small extracellular vesicles, which are an important therapeutic component of MSCs, have been reported to be effective in a mouse model of BPD. However, the affected cell types and detailed underlying mechanisms are unclear. In this study, we found that human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) were successfully absorbed by lung tissue after intratracheal administration, and remained in the lungs for at least 72 h. The results showed that hucMSC-sEVs restored alveolar structure and lung function, and ameliorated pulmonary hypertension in a rat model of BPD. The number of Ki-67-positive lung cells were improved, while the number of TUNEL-positive lung cells were reduced in our hucMSC-sEV-treated BPD model. Additionally, SP-C staining (a marker of type II alveolar epithelial cells, TIIAECs) and CD31 staining (a marker of pulmonary vascular endothelial cells, PVECs) were both increased in a hyperoxia-induced BPD model treated with hucMSC-sEVs. In vitro, under hyperoxic conditions, the tube-like structure formation was improved in human umbilical vein endothelial cells, and the proliferation was increased and the apoptosis was attenuated in MLE-12 cells treated with hucMSC-sEVs. Furthermore, we observed downregulated expression of PTEN and cleaved-caspase3, and upregulated expression of p-Akt and vascular endothelial growth factor-A in our hucMSC-sEV-treated BPD model. In conclusion, hucMSC-sEVs improved alveolarization and angiogenesis in a rat BPD model by protecting TIIAECs and PVECs, which were associated with the PTEN/Akt signaling pathway.

Single cell RNA sequencing identifies transcriptional signature of atherosusceptible endothelium of aorta in diabetes associated atherosclerosis

Abstract Background Transcriptomic analyses have provided invaluable information for linking genotypes to phenotypes. However, despite the near identical genotype, each cell types in our body has a unique gene expression signature. Deep sequencing at single cell resolution has provided a unique opportunity to unbiasedly discover cellular heterogeneity, disease associated cell populations and to characterise the cell specific transcriptomic landscape. Cardiovascular (CV) disease, a leading cause of death worldwide, is caused mainly by atherosclerosis, a pathological build-up of plaque within arterial vessel walls. Fluid mechanical forces generated by disturbed blood flow are long known to cause structural and transcriptional changes in the vascular endothelium. Atherosclerosis develops near branches and bends of arteries exposed to disturbed blood flow. Diabetes accelerates atherosclerosis development and indeed, represents an independent risk factor. However, the transcriptional signature of atheroprone endothelium in the diabetic aorta has not been investigated previously for this CV complication. Purpose This study was designed to apply a single cell RNA sequencing (scRNA-seq) approach to identify the transcriptional signature of atherosusceptible endothelium in diabetes associated atherosclerosis. Methods Diabetes was induced with streptozotocin in ApoEs−/− mice and followed for 10 weeks. Cells from digested aortae of control and diabetic mice were FACS-sorted for viable and metabolically active cells. These cells were loaded onto the Chromium Single Cell Controller (10X Genomics) to generate a single cell and gel bead emulsion. ScRNA-seq libraries were prepared with Single Cell 3' Solution V2 kit (10X Genomics) and sequenced with Illumina Nova-seq 6000. We have applied scRNA-seq to identify atheroprone endothelial cells in the aorta. Results and conclusion The atheroprone endothelial cells show distinct transcriptional profile with more than six hundred genes differentially expressed. ScRNA-seq allowed us not only to distinguish the two transcriptionally distinct endothelial subpopulations but also to identify a diabetes associated gene expression signature unique to atheroprone endothelial cells as compared to all other cell types in the aorta. We identified seventeen genes uniquely dysregulated in the diabetic atheroprone endothelial cell (Cut off = FDR s&amp;lt;0.05, Fold change at least 2-fold). This includes Protein C receptor (Procr) which has recently been identified as a marker for blood vascular endothelial stem cells (VESCs). Downregulation of Procr in the atheroprone endothelial cells of diabetic aorta as identified in our scRNA-seq data indicates that diabetes may limit the vascular repair by targeting VESCs thus contributing to accelerated atherosclerosis. These exciting novel findings have uncovered the transcriptomic landscape of atherosusceptible endothelium of aorta at the single cell level as seen in diabetes associated atherosclerosis. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Heart Foundation of Australia; NHMRC National Health and Medical Research Council of Australia