CD146+ Pericytes Subset Isolated from Human Micro-Fragmented Fat Tissue Display a Strong Interaction with Endothelial Cells: A Potential Cell Target for Therapeutic Angiogenesis

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2⁺/PDGFRβ⁺/CD105⁺ cells. Here, we tested the fat-derived PCs for the dispensability of the CD146 marker with the aim of better understanding the role of these PC subpopulations on angiogenesis. Cells from both CD146-positive (CD146⁺) and negative (CD146⁻) populations were observed to interact with human umbilical vein ECs (HUVECs). In addition, fat-derived PCs were able to induce angiogenesis of ECs in spheroids assay; and conditioned medium (CM) from both PCs and fat tissue itself led to the proliferation of ECs, thereby marking their role in angiogenesis stimulation. However, we found that CD146⁺ cells were more responsive to PDGF-BB-stimulated migration, adhesion, and angiogenic interaction with ECs, possibly owing to their higher expression of NCAM/CD56 than the corresponding CD146⁻ subpopulation. We conclude that in fat tissue, CD146-expressing cells may represent a more mature pericyte subpopulation that may have higher efficacy in controlling and stimulating vascular regeneration and stabilization than their CD146-negative counterpart.     

Angiogenic Effects and Crosstalk of Adipose-Derived Mesenchymal Stem/Stromal Cells and Their Extracellular Vesicles with Endothelial Cells

Adipose-derived mesenchymal stem/stromal cells (ASCs) are an adult stem cell population able to self-renew and differentiate into numerous cell lineages. ASCs provide a promising future for therapeutic angiogenesis due to their ability to promote blood vessel formation. Specifically, their ability to differentiate into endothelial cells (ECs) and pericyte-like cells and to secrete angiogenesis-promoting growth factors and extracellular vesicles (EVs) makes them an ideal option in cell therapy and in regenerative medicine in conditions including tissue ischemia. In recent angiogenesis research, ASCs have often been co-cultured with an endothelial cell (EC) type in order to form mature vessel-like networks in specific culture conditions. In this review, we introduce co-culture systems and co-transplantation studies between ASCs and ECs. In co-cultures, the cells communicate via direct cell–cell contact or via paracrine signaling. Most often, ASCs are found in the perivascular niche lining the vessels, where they stabilize the vascular structures and express common pericyte surface proteins. In co-cultures, ASCs modulate endothelial cells and induce angiogenesis by promoting tube formation, partly via secretion of EVs. In vivo co-transplantation of ASCs and ECs showed improved formation of functional vessels over a single cell type transplantation. Adipose tissue as a cell source for both mesenchymal stem cells and ECs for co-transplantation serves as a prominent option for therapeutic angiogenesis and blood perfusion in vivo.     

Specific Local Predictors That Reflect the Tropism of Endometriosis—A Multiple Immunohistochemistry Technique

Ectopic endometrial epithelium associates a wide spectrum of symptomatology. Their evolution can be influenced by inflammatory and vascular changes, that affect not only the structure and cell proliferation rate, but also symptoms. This prospective study involved tissue samples from surgically treated patients, stained using classical histotechniques and immunohistochemistry. We assessed ectopic endometrial glands (CK7+, CK20−), adjacent blood vessels (CD34+), estrogen/progesterone hormone receptors (ER+, PR+), inflammatory cells (CD3+, CD20+, CD68+, Tryptase+), rate of inflammatory cells (Ki67+) and oncoproteins (BCL2+, PTEN+, p53+) involved in the development of endometriosis/adenomyosis. A CK7+/CK20− expression profile was present in the ectopic epithelium and differentiated it from digestive metastases. ER+/PR+ were present in all cases analyzed. We found an increased vascularity (CD34+) in the areas with abdominal endometriosis and CD3+−:T-lymphocytes, CD20+−:B-lymphocytes, CD68+:macrophages, and Tryptase+: mastocytes were abundant, especially in cases with adenomyosis as a marker of proinflammatory microenvironment. In addition, we found a significantly higher division index-(Ki67+) in the areas with adenomyosis, and inactivation of tumor suppressor genes-p53+ in areas with neoplastic changes. The inflammatory/vascular/hormonal mechanisms trigger endometriosis progression and neoplastic changes increasing local pain. Furthermore, they may represent future therapeutic targets. Simultaneous-multiple immunohistochemical labelling represents a valuable technique for rapidly detecting cellular features that facilitate comparative analysis of the studied predictors.     

4-Pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR)—A Novel Oncometabolite Modulating Cancer-Endothelial Interactions in Breast Cancer Metastasis

The accumulation of specific metabolic intermediates is known to promote cancer progression. We analyzed the role of 4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR), a nucleotide metabolite that accumulates in the blood of cancer patients, using the 4T1 murine in vivo breast cancer model, and cultured cancer (4T1) and endothelial cells (ECs) for in vitro studies. In vivo studies demonstrated that 4PYR facilitated lung metastasis without affecting primary tumor growth. In vitro studies demonstrated that 4PYR affected extracellular adenine nucleotide metabolism and the intracellular energy status in ECs, shifting catabolite patterns toward the accumulation of extracellular inosine, and leading to the increased permeability of lung ECs. These changes prevailed over the direct effect of 4PYR on 4T1 cells that reduced their invasive potential through 4PYR-induced modulation of the CD73-adenosine axis. We conclude that 4PYR is an oncometabolite that affects later stages of the metastatic cascade by acting specifically through the regulation of EC permeability and metabolic controls of inflammation.     

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies

Endothelial cells (ECs) lining the blood vessels are important players in many biological phenomena but are crucial in hypoxia-dependent diseases where their deregulation contributes to pathology. On the other hand, processes mediated by ECs, such as angiogenesis, vessel permeability, interactions with cells and factors circulating in the blood, maintain homeostasis of the organism. Understanding the diversity and heterogeneity of ECs in different tissues and during various biological processes is crucial in biomedical research to properly develop our knowledge on many diseases, including cancer. Here, we review the most important aspects related to ECs’ heterogeneity and list the available in vitro tools to study different angiogenesis-related pathologies. We focus on the relationship between functions of ECs and their organo-specificity but also point to how the microenvironment, mainly hypoxia, shapes their activity. We believe that taking into account the specific features of ECs that are relevant to the object of the study (organ or disease state), especially in a simplified in vitro setting, is important to truly depict the biology of endothelium and its consequences. This is possible in many instances with the use of proper in vitro tools as alternative methods to animal testing.     

MiR-126-3p Is Dynamically Regulated in Endothelial-to-Mesenchymal Transition during Fibrosis

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFβ2 and IL1β. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.     

The Adaptive Immune Landscape of the Colorectal Adenoma–Carcinoma Sequence

Background. The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma–carcinoma sequence (ACS). Methods. We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). Results. The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. Conclusions. The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.     

Studies on anti-tumor and antimetastatic activities of fullerenol in a mouse breast cancer model

The purpose was to examine the anti-tumor and antimetastatic activities of fullerenol and their related mechanisms. Thirty EMT-6 tumor-bearing mice were injected intraperitoneally with 0.1 ml saline or 0.1 ml saline containing fullerenol C₆₀(OH)₂₀ (0.08 and 0.4 mg/ml) daily for 16 days. Using tumor tissues, we investigated imbalances in the oxidative defense system and the expression of several angiogenesis factors. C₆₀(OH)₂₀ exhibits anti-tumor and antimetastatic activities in EMT-6 breast cancer metastasis model. Treatment with C₆₀(OH)₂₀ was found to modulate oxidative stress significantly. The expression of several angiogenesis factors was reduced in tumor tissues after treatment with fullerenol. Importantly, CD31 (also known as PECAM-1, platelet endothelial cell adhesion molecule) expression and vessel density were markedly reduced in tumors from fullerenol-treated mice compared with controls. Modulation of oxidative stress in tumor tissues, inhibition of the formation of angiogenesis factors, and subsequent reduction in tumor vessel density and the nutrient supply to tumor cells could be important mechanisms by which fullerenol aggregates inhibit tumor growth and suppress carcinoma metastasis in vivo.     

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.     

Suppression of Lipid Accumulation by Indole-3-Carbinol Is Associated with Increased Expression of the Aryl Hydrocarbon Receptor and CYP1B1 Proteins in Adipocytes and with Decreased Adipocyte-Stimulated Endothelial Tube Formation

This study investigated the effects of indole-3-carbinol (I3C) on adipogenesis- and angiogenesis-associated factors in mature adipocytes. The cross-talk between mature adipocytes and endothelial cells (ECs) was also explored by cultivating ECs in a conditioned medium (CM) by using I3C-treated adipocytes. The results revealed that I3C significantly inhibited triglyceride accumulation in mature adipocytes in association with significantly increased expression of AhR and CYP1B1 proteins as well as slightly decreased nuclear factor erythroid-derived factor 2–related factor 2, hormone-sensitive lipase, and glycerol-3-phosphate dehydrogenase expression by mature adipocytes. Furthermore, I3C inhibited CM-stimulated endothelial tube formation, which was accompanied by the modulated secretion of angiogenic factors in adipocytes, including vascular endothelial growth factor, interleukin-6, matrix metalloproteinases, and nitric oxide. In conclusion, I3C reduced lipid droplet accumulation in adipocytes and suppressed adipocyte-stimulated angiogenesis in ECs, suggesting that I3C is a potential therapeutic agent for treating obesity and obesity-associated disorders.     

Rho GTPases in Retinal Vascular Diseases

The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell–cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.     

Changes in Immunogenicity during the Development of Urinary Bladder Cancer: A Preliminary Study

In the present study, we evaluated tumor-infiltrating lymphocytes (TILs) and blood regulatory T lymphocyte (Tregs, CD4+/CD25+/FoxP3+) expression in bladder cancer patients. The number of CD4+, CD8+, CD25+, FoxP3+ and CD20+ TILs was analyzed in association with clinico-pathomorphological features. In more advanced metastasizing tumors, showing non-classic differentiation (ND) and a more aggressive tissue invasion type (TIT), the number of TILs decreased. A low number of CD4+ TILs was associated with poor prognosis. Similarly, Treg frequency before surgery and after surgical treatment was significantly lower in more advanced tumors. The changes in TILs, as well as of local and systemic Tregs, were accompanied by changes in the histological phenotype of urothelial carcinoma regarding pT stage, NDs, TIT, and clinical outcomes. The number of TILs and the frequency of blood Tregs (indicators of antitumor response) may be essential for choosing an immunotherapy that is adjusted to the immune status according to the phase of tumor growth. Moreover, a significant reduction in the number of CD4+ and CD8+ TILs with the development of NDs in more advanced tumors may be associated with lower tumor immunogenicity, resulting in immune tolerance towards tumor tissue. These observations and the tendency of urothelial bladder carcinoma to undergo NDs in a heterogeneous manner during tumor progression suggest complex interactions between bladder cancer immunogenicity and stages of tumor progression.     

Vascular Endothelial Growth Factor,a Key Modulator of the Anti-Tumor Immune Response

During tumor growth, angiogenesis is required to ensure oxygen and nutrient transport to the tumor. Vascular endothelial growth factor (VEGF) is the major inducer of angiogenesis and appears to be a key modulator of the anti-tumor immune response. Indeed, VEGF modulates innate and adaptive immune responses through direct interactions and indirectly by modulating protein expressions on endothelial cells or vascular permeability. The inhibition of the VEGF signaling pathway is clinically approved for the treatment of several cancers. Therapies targeting VEGF can modulate the tumor vasculature and the immune response. In this review, we discuss the roles of VEGF in the anti-tumor immune response. In addition, we summarize therapeutic strategies based on its inhibition, and their clinical approval.     

Short Hairpin RNA (shRNA) Ether �� go-go 1 (Eag1) Inhibition of Human Osteosarcoma Angiogenesis via VEGF/PI3K/AKT Signaling

Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers but the therapeutic potential of Eag1 in osteosarcoma remains elusive. In this study, we constructed an Ad5-Eag1-shRNA vector and evaluated its efficiency for Eag1 knockdown and its effects on osteosarcoma. Our results showed that Ad5-Eag1-shRNA had high interference efficiency of Eag1 expression and suppressed osteosarcoma growth both in vitro and in vivo. To explore the molecular mechanism underlying tumor growth inhibition induced by Eag1 silencing, the intratumoral microvessel density (MVD) was assessed by CD31 staining and the expression of vascular endothelial growth factor (VEGF) was detected by Western blot analysis. We found that Eag1 silencing led to decreased angiogenesis and VEGF expression in the xenograft model of osteosarcoma. Finally, we detected a time-dependent decrease in VEGF expression and considerably reduced phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation in osteosarcoma cells treated by Eag1 shRNA. Taken together, our results suggest that Eag1 silencing inhibits tumor growth and angiogenesis in osteosarcoma via the down regulation of VEGF/PI3K/AKT signaling.     

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.     

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts,Keratinocytes, and Endothelial Cells

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5′-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5′-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.     

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs’ size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 μm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 μm EVs contribute to the regulation of NTHY gene expression and angiogenesis.     

SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate–SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate–SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.