Pro-angiogenic Molecules Research Articles

Soluble CD146 cooperates with VEGF-A to generate an immunosuppressive microenvironment in CD146-positive tumors: interest of a combined antibody-based therapy.

Tumor development necessitates immune escape through different mechanisms. To counteract these effects, the development of therapies targeting Immune Checkpoints (ICP) has generated interest as they have produced lasting objective responses in patients with advanced metastatic tumors. However, many tumors do not respond to inhibitors of ICP, necessitating to further study the underlying mechanisms of exhaustion. Vascular Endothelial Growth Factor a (VEGFa), a pro-angiogenic molecule secreted by tumors, was described to participate to tumor immune exhaustion by increasing ICP, justifying in part the use of an anti-VEGFa monoclonal antibody (mAb), bevacizumab, in patients. However, recent studies from our group have demonstrated that tumors can escape anti-VEGFa therapy through the secretion of soluble CD146 (sCD146). In this study, we show that both VEGFa and sCD146 cooperate to create an immunosuppressive microenvironment by increasing the expression of ICP. In addition, sCD146 favors pro-tumoral M2-type macrophages and induces the secretion of pro-inflammatory cytokines. An anti-sCD146 mAb reverses these effects and displays additive effects with anti-VEGFa antibody to eliminate tumors in a syngeneic murine model grafted with melanoma cells. Combining bevacizumab with mucizumab could thus be of major therapeutic interest to prevent immune escape in malignant melanoma and other CD146-positive tumors.

Wolbachia Promotes an Anti-Angiogenic Response Using an In Vitro Model of Vascular Endothelial Cells in Relation to Heartworm Disease.

Heartworm disease caused by Dirofilaria immitis is a vector-borne zoonotic disease responsible for the infection of mainly domestic dogs and cats, or these are those for which the most data are known. Humans are an accidental host where a benign, asymptomatic pulmonary nodule may originate. Dirofilaria immitis also harbours the endosymbiont bacteria of the genus Wolbachia, which play a role in moulting, embryogenesis, inflammatory pathology, and immune response. When Wolbachia sp. is released into the bloodstream, endothelial and pulmonary damage is exacerbated, further encouraging thrombus formation and pulmonary hypertension, facilitating congestive heart failure and death of the animal. Previous studies have shown that parasite excretory/secretory products are able to activate the pro-angiogenic pathway (formation of new vessels) to facilitate parasite survival. The aim of this study was to analyse the role of Wolbachia sp. and its relationship with the cellular processes and the angiogenic pathway in a model of human endothelial cells in vitro. The use of recombinant Wolbachia Surface Protein (rWSP) showed that its stimulation exerted an anti-angiogenic effect by detecting an increase in the production of VEGFR-1/sFlt1 and sEndoglin and did not affect the production of VEGFR-2 and mEndoglin (pro-angiogenic molecules). Furthermore, it did not stimulate cell proliferation or migration, although it did negatively stimulate the formation of pseudocapillaries, slowing down this process. These cellular processes are directly related to the angiogenic pathway so, with these results, we can conclude that Wolbachia sp. is related to the stimulation of the anti-angiogenic pathway, not facilitating the survival of D. immitis in vascular endothelium.

Apoptotic Extracellular Vesicles from Supernumerary Tooth-Derived Pulp Stem Cells Transfer COL1A1 to Promote Angiogenesis via PI3K/Akt/VEGF Pathway.

Angiogenesis is a tightly controlled process that initiates the formation of new vessels and its dysfunction can lead to life-threatening diseases. Apoptotic extracellular vesicles (ApoEVs) have emerged as a proangiogenic agent with high safety and isolation efficiency profile, and ApoEVs from supernumerary tooth-derived pulp stem cells (SNTSC-ApoEVs) have their unique advantages with an easily accessible parental cell source and non-invasive cell harvesting. However, the detailed characteristics of SNTSC-ApoEVs are largely unknown. This study aimed to investigate the proangiogenic capacity and function molecule of SNTSC-ApoEVs. SNTSC-ApoEVs were isolated and characterized. In vitro effects of SNTSC-ApoEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, wound healing, transwell, and tube formation assays. The mRNA and protein levels of proangiogenic genes were quantified by qRT-PCR, Western blot, and immunofluorescence analysis. A Matrigel plug model was established in 6-week-old male nu/nu mice for one week, and the in vivo impact of SNTSC-ApoEVs on micro-vessel formation was assessed by histological analysis. Proteomic analysis and RNA sequencing were performed to explore the active ingredients and underlying mechanisms. SNTSC-ApoEVs enhanced the proliferation, migration, and angiogenesis of HUVECs in vitro. In the Matrigel plug model in vivo, SNTSC-ApoEVs promoted CD31-positive luminal structure formation. Apart from expressing general ApoEV markers, SNTSC-ApoEVs were enriched with multiple proteins related to extracellular matrix-cell interactions. Mechanistically, SNTSC-ApoEVs transferred COL1A1 to HUVECs and promoted endothelial functions by activating the PI3K/Akt/VEGF cascade. SNTSC-ApoEVs can promote angiogenesis by transferring the functional molecule COL1A1 and activating the PI3K/Akt/VEGF pathway, making SNTSC-ApoEVs a promising strategy for the treatment of angiogenesis-related diseases.

Metastasis and angiogenesis in cervical cancer: key aspects of purinergic signaling in platelets and possible therapeutic targets.

Cervical cancer ranks as the fourth most common and fatal cancer among women worldwide. Studies have demonstrated a strong association between purinergic platelet signaling and tumor progression in this type of cancer. The literature shows that neoplastic cells, when in the bloodstream, secrete adenosine triphosphate (ATP) and adenosine nucleotide diphosphate (ADP) that act on their corresponding platelet P2Y and P2X receptors. The interaction of these nucleotides with their receptors results in platelet activation and degranulation, ensuing several consequences, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor, matrix metalloproteinases, ADP, and ATP. These molecules play essential roles in angiogenesis and tumor metastasis in cervical cancer. Several purinergic receptors are found in endothelial cells. Their activation, especially P2Y2, by the nucleotides released by platelets can induce relaxation of the endothelial barrier and consequent extravasation of tumor cells, promoting the development of metastases. Cancer cells that enter the bloodstream during the metastatic process are also subject to high shear stress and immune surveillance. In this context, activated platelets bind to circulating tumor cells and protect them against shear stress and the host's immune system, especially against natural killer cells, facilitating their spread throughout the body. Furthermore, activation of the P2Y12 receptor present on the platelet surface promotes the release of VEGF, the main inducer of angiogenesis in cervical cancer, in addition to increasing the concentration of several other pro-angiogenic molecules. Therefore, this review will address the role of platelet purinergic signaling in tumor progression of cervical cancer and propose possible therapeutic targets.

The Application of Umbilical Cord Blood-derived Platelet Gel for Skin Ulcers Associated With Chronic Graft-Versus-Host Disease in Pediatrics: A Randomized Trial

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative strategy against a variety of malignant and nonmalignant disorders. However, acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively) commonly complicate this approach, culminating in substantial morbidities and mortalities. The integumentary system is the preponderant organ involved in cGVHD, and its response to existing treatments, including well-versed immunosuppressants and novel targeted therapies, is not desirable. Despite the rarity, ulcers of sclerotic skin cGVHD are treatment-refractory and associated with significant morbidities and an exaggerated risk of infectious complications. Platelet-rich plasma (PRP) and its derivatives are endowed with growth factors and proangiogenic molecules and hold regenerative potential. This study aimed to assess the safety and efficacy of the application of platelet gel-containing dressing against ulcerative skin cGVHD in pediatric patients. This randomized trial is conducted at the hematopoietic stem cell transplantation unit of the Children's Medical Center Hospital in Tehran, Iran. Twenty-one pediatric patients (aged between 5 and 15 years) were initially enrolled, and 16 met the inclusion criteria. All cases (4 females) were recipients of allo-HSCT who had been complicated with symmetrically or near-symmetrically ulcerative sclerotic skin cGVHD. Fresh umbilical cord blood (UCB) was obtained from healthy donors and underwent centrifugation using a novel PRP preparation kit in a single-step process. Platelet gel was produced by adding thrombin to the isolated buffy coat layer. Two similar ulcers of each patient were randomized to receive either conventional dressing or platelet gels up to 6 times. At each time point evaluation, ulcer size and its relative reduction compared to the basal size were recorded. Included patients received a total of 80 platelet gel-containing dressings. While the mean sizes of randomized ulcers at the beginning of the study were similar, their differences became significant 15 days after the initiation of intervention (P = .019). In addition, the mean reduction in the ulcers’ surface area (in comparison to their baseline values) was significantly higher for the intervention arm at all evaluation points (P = .001 for day 5 and P < .001 for subsequent time points). At the end of the trial, the number of ulcers with a more than 50% reduction in size was 14 (87.5%) in the intervention arm (including 6 completely healed ulcers) versus 1 (6.25%, which was not completely healed) in the control arm (P < .001). None of the patients exhibited any localized or systemic treatment-related adverse events. In this study, using a relatively large number of cases, we showed that UCB-derived platelet gel is a safe, feasible, and effective curative approach for skin ulcers of sclerotic skin cGVHD in pediatric patients. Designing upcoming trials on the efficacy of this therapeutic approach for ocular, mucosal, and acute skin GVHD is prudent. Retrospectively registered at the Iranian Registry of Clinical Trials (registration number IRCT20190101042197N1) on August 24, 2020.

The Association Between Epidermal Growth Factor rs3756261 A/G Gene Polymorphism and the Risk of Ankylosing Spondylitis in a Chinese Han Population.

Epidermal growth factor (EGF) is a potent pro-angiogenic molecule promoting the angiogenic phenotype of ankylosing spondylitis (AS). Studies demonstrated that EGF rs3756261 polymorphism was associated with the risk of inflammatory diseases, but not including AS. To investigate the association between EGF rs3756261 polymorphism and the risk of AS, we genotyped the EGF rs3756261 polymorphism in 208 patients with AS and 412 controls in a Chinese Han population using a custom-by-design 48-Plex SNP scanTM Kit. The serum EGF levels were measured using an enzyme-linked immunosorbent assay in 208 AS patients and 412 controls. Our data indicated that EGF rs3756261 polymorphism was associated with an increased risk of AS in the Chinese Han population. Stratified analyses indicated that the EGF rs3756261 polymorphism elevated the risk of AS among the males, smokers, drinkers and those aged <30 years. In addition, the EGF rs3756261 polymorphism was related to increased CRP and HLA-B27 levels in AS patients. Next, we found that the average serum levels of EGF were significantly higher in AS patients compared with controls. Meanwhile, EGF serum levels were significantly higher in AG genotype carriers when compared with AA genotype carriers in AS patients. In conclusion, this study indicated that EGF rs3756261 polymorphism was associated with the risk of AS and EGF serum levels in a Chinese Han population.

Spheroid size influences cellular senescence and angiogenic potential of mesenchymal stromal cell-derived soluble factors and extracellular vesicles.

Introduction: The secretome of mesenchymal stromal cells (MSCs) serves as an innovative tool employed in the regenerative medicine approach. In this particular context, three-dimensional (3D) culture systems are widely utilized to better replicate in vivo conditions and facilitate prolonged cell maintenance during culture. The use of spheroids enables the preservation of the classical phenotypical characteristics of MSCs. However, the distinct microenvironment within the spheroid may impact the secretome, thereby enhancing the angiogenic properties of adult MSCs that typically possess a reduced angiogenic potential compared to MSCs derived from perinatal tissues due to the hypoxia created in the internal region of the spheroid. Methods: In this study, large spheroids (2,600 cells, ∼300μm diameter) and small spheroids (1,000 cells, ∼200μm diameter) were used to examine the role of spheroid diameter in the generation of nutrients and oxygen gradients, cellular senescence, and the angiogenic potential of secreted factors and extracellular vesicles (EVs). Results: In this study, we demonstrate that large spheroids showed increased senescence and a secretome enriched in pro-angiogenic factors, as well as pro-inflammatory and anti-angiogenic cytokines, while small spheroids exhibited decreased senescence and a secretome enriched in pro-angiogenic molecules. We also demonstrated that 3D culture led to a higher secretion of EVs with classical phenotypic characteristics. Soluble factors and EVs from small spheroids exhibited higher angiogenic potential in a human umbilical vein endothelial cell (HUVEC) angiogenic assay. Discussion: These findings highlighted the necessity of choosing the appropriate culture system for obtaining soluble factors and EVs for specific therapeutic applications.

Association analysis of Vascular Endothelial Growth Factor-A (VEGF-A) polymorphism in rheumatoid arthritis using computational approaches

Rheumatoid arthritis (RA), is marked by joint inflammation leading to pannus formation which results in cartilage destruction promoting bone erosion. The pathological hallmark of RA includes synovial hyperplasia and synovial angiogenesis. Active tissue neovascularization is observed in RA. Vascular endothelial Growth factor A (VEGFA), an endothelial cell-specific proangiogenic molecule is triggered by hypoxic cells and its levels are upregulated in RA. The aim of this study was to investigate functional and pathogenic VEGFA variants and to identify the impact of point mutation in VEGFA’s interaction with VEGFR2 and how these polymorphisms affect the susceptibility and severity of RA. We investigated impact of these point mutations on the stability of VEGFA using various computational tools. These mutations were further identified by conservational profile as they are highly involved as structural and functional mutations. Furthermore, these selected variants were modelled and docked against targeted domain regions IGD2 and IGD3 of VEGFR2. Further molecular dynamic simulations were performed using Gromacs. Out of 168 nsSNPS, 19 were highlighted as highly pathogenic using insilico prediction tools. InterPro and ConSurf revealed domains and conserved variants respectively. After stability analysis, we concluded that almost all the mutations were responsible for decreasing the protein stability. HOPE predicted that all the selected damaging nsSNPs were present in the domain which is essential for the functioning of VEGFA protein. Constructed Ramachandran plot and ERRAT validated the quality of all the models. Based on the interactions predicted by STRING database, we performed Protein–Protein docking between VEGFA and VEGFR2. We found few conserved interactions and new polar contacts among wild-type and mutants with VEGFR2. From the simulations, we concluded that mutant R108Q was the most stabilizing mutant among all others whereas R82Q, C86Y, and R108W complexed with VEGFR2 were comparatively less stabilizing as compared to the wild type. This study provides insight into pathogenic nsSNPs that can affect VEGFA protein structure and function. These high-risk variants must be taken into consideration for genetic screening of patients suffering from RA.

Signalling pathways associated with impaired angiogenesis in Alzheimer’s Disease

AbstractBackgroundBrain perfusion and blood brain barrier (BBB) integrity are reduced in Alzheimer’s disease (AD). We hypothesized that b‐amyloid (Aβ) induces dysfunction of pathways in vascular cells that regulate angiogenesis.MethodWe performed single nucleus RNA sequencing (snRNAseq) of vascular cells isolated from post mortem samples from multiple cortical regions from AD patients (Braak V‐VI) and non‐diseased controls (NDC, Braak 0‐II). We developed a meta‐analytic approach to integrate these data with related publicly available datasets. We performed differential gene expression (DGE) analyses as a categorical comparison of AD vs NDC and as a confound‐controlled regression of transcriptomic alterations by regional total Aβ. We also performed cell‐specific gene co‐expression analyses identify modules differentially expressed in AD relative to NDC.ResultOur results describe differential expression of genes central to pathways sustaining angiogenesis, proliferation and migration of endothelial cells and the integrity of the BBB with either AD or greater pathological protein load. HIF1A and FGF2, proangiogenic molecules which both induce VEGF, are upregulated in EC, consistent the parenchymal hypoxic response shown repeatedly in prior studies. However, components of signalling pathways downstream of VEGFR2 are downregulated, suggesting a pathological block to angiogenesis with greater Aβ load. While the proangiogenic stimuli promote the expression of genes such as PPP3CA and ITPR1, coding for calcineurin, which contribute to VEGF signalling through the PLCγ‐Ca2+ signalling pathway are upregulated in AD, expression of RAC1 and RHOJ, which enable cellular migration and adhesion, is downregulated. While ANGPT2 expression is also upregulated, the angiopoietin receptor (TIE2, coded by TEK) is downregulated. Downregulation of ADAM10 in AD and upregulation of other NOTCH pathway genes with greater Aβ load suggests altered NOTCH signalling.ConclusionOverall, a hypoxic tissue environment in AD is accompanied by dysfunctional angiogenesis related to Aβ load. Evidence for multiple sites of dysfunction were identified, involving impaired angiopoietin responses, vascular cell migration and NOTCH signaling.

Adipose-Derived Stem Cells to Treat Ischemic Diseases: The Case of Peripheral Artery Disease

Critical limb ischemia incidence and prevalence have increased over the years. However, there are no successful treatments to improve quality of life and to reduce the risk of cardiovascular and limb events in these patients. Advanced regenerative therapies have focused their interest on the generation of new blood vessels to repair tissue damage through the use of stem cells. One of the most promising sources of stem cells with high potential in cell-based therapy is adipose-derived stem cells (ASCs). ASCs are adult mesenchymal stem cells that are relatively abundant and ubiquitous and are characterized by a multilineage capacity and low immunogenicity. The proangiogenic benefits of ASCs may be ascribed to: (a) paracrine secretion of proangiogenic molecules that may stimulate angiogenesis; (b) secretion of microvesicles/exosomes that are also considered as a novel therapeutic prospect for treating ischemic diseases; and (c) their differentiation capability toward endothelial cells (ECs). Although we know the proangiogenic effects of ASCs, the therapeutic efficacy of ASCs after transplantation in peripheral artery diseases patients is still relatively low. In this review, we evidence the potential therapeutic use of ASCs in ischemic regenerative medicine. We also highlight the main challenges in the differentiation of these cells into functional ECs. However, significant efforts are still needed to ascertain relevant transcription factors, intracellular signaling and interlinking pathways in endothelial differentiation.

Modulation of angiogenic switch in reprogramming browning and lipid metabolism in white adipocytes

Thermogenic activation via trans-and de novo browning of white adipocytes is a promising strategy to accelerate lipid metabolism for regulating obesity-related disorders. In this study, we investigated the intricate interplay between angiogenic regulation and browning in white adipocytes using the bioactive compound, resveratrol (Rsv). Rsv has previously been documented for its regulatory influence on the trans and de novo browning of white adipocytes. Our findings revealed that concurrent activation of angiogenesis is prerequisite for inducing browning within the microenvironment of white adipocytes when exposed to browning activators. Additionally, we observed a significant browning effect on white adipocytes when the local adipose tissue environment was prompted to undergo angiogenesis, notably facilitated by a proangiogenic molecule known as Vascular endothelial growth factor (VEGF). Intriguingly, this effect was reversed when angiogenesis was inhibited by treatment with the antiangiogenic agent thalidomide. Furthermore, the study revealed the role of VEGF in paracrine activation of white adipocytes resulting in the induction of browning in both 3T3-L1 cell lines and primary mouse white adipocytes. The cross-talk between angiogenesis and browning was found to be initiated via the transcriptional activation of Estrogen receptor α (ERα) triggering the VEGF/VEGFR2 signaling pathway leading to browning and a reconfiguration of lipid metabolism within adipocytes. In conclusion, this study sheds light on the intricate cross-talk between angiogenesis and browning of white adipocytes. Notably, the findings underscore the reciprocal relationship between these processes, wherein inhibition of one process exerts discernible effects on the other.

Sulfated oligosaccharide activates endothelial Notch for inducing macrophage-associated arteriogenesis to treat ischemic diseases.

Ischemic diseases lead to considerable morbidity and mortality, yet conventional clinical treatment strategies for therapeutic angiogenesis fall short of being impactful. Despite the potential of biomaterials to deliver pro-angiogenic molecules at the infarct site to induce angiogenesis, their efficacy has been impeded by aberrant vascular activation and off-target circulation. Here, we present a semisynthetic low-molecular sulfated chitosan oligosaccharide (SCOS) that efficiently induces therapeutic arteriogenesis with a spontaneous generation of collateral circulation and blood reperfusion in rodent models of hind limb ischemia and myocardial infarction. SCOS elicits anti-inflammatory macrophages' (Mφs') differentiation into perivascular Mφs, which in turn directs artery formation via a cell-to-cell communication rather than secretory factor regulation. SCOS-mediated arteriogenesis requires a canonical Notch signaling pathway in Mφs via the glycosylation of protein O-glucosyltransferases 2, which results in promoting arterial differentiation and tissue repair in ischemia. Thus, this highly bioactive oligosaccharide can be harnessed to direct efficiently therapeutic arteriogenesis and perfusion for the treatment of ischemic diseases.

Application of hypoxia-mesenchymal stem cells in treatment of anaerobic bacterial wound infection: wound healing and infection recovery.

Mesenchymal stromal cells, commonly referred to as MSCs, are a type of multipotent stem cells that are typically extracted from adipose tissue and bone marrow. In the field of tissue engineering and regenerative medicine, MSCs and their exosomes have emerged as revolutionary tools. Researchers are now devoting greater attention to MSCs because of their ability to generate skin cells like fibroblasts and keratinocytes, as well as their distinctive potential to decrease inflammation and emit pro-angiogenic molecules at the site of wounds. More recent investigations revealed that MSCs can exert numerous direct and indirect antimicrobial effects that are immunologically mediated. Collectively, these antimicrobial properties can remove bacterial infections when the MSCs are delivered in a therapeutic setting. Regardless of the positive therapeutic potential of MSCs for a multitude of conditions, transplanted MSC cell retention continues to be a major challenge. Since MSCs are typically administered into naturally hypoxic tissues, understanding the impact of hypoxia on the functioning of MSCs is crucial. Hypoxia has been postulated to be among the factors determining the differentiation of MSCs, resulting in the production of inflammatory cytokines throughout the process of tissue regeneration and wound repair. This has opened new horizons in developing MSC-based systems as a potent therapeutic tool in oxygen-deprived regions, including anaerobic wound infection sites. This review sheds light on the role of hypoxia-MSCs in the treatment of anaerobic bacterial wound infection in terms of both their regenerative and antimicrobial activities.

The Inhibition of the FGFR/PI3K/Akt Axis by AZD4547 Disrupts the Proangiogenic Microenvironment and Vasculogenic Mimicry Arising from the Interplay between Endothelial and Triple-Negative Breast Cancer Cells.

Vasculogenic mimicry (VM), a process in which aggressive cancer cells form tube-like structures, plays a crucial role in providing nutrients and escape routes. Highly plastic tumor cells, such as those with the triple-negative breast cancer (TNBC) phenotype, can develop VM. However, little is known about the interplay between the cellular components of the tumor microenvironment and TNBC cells' VM capacity. In this study, we analyzed the ability of endothelial and stromal cells to induce VM when interacting with TNBC cells and analyzed the involvement of the FGFR/PI3K/Akt pathway in this process. VM was corroborated using fluorescently labeled TNBC cells. Only endothelial cells triggered VM formation, suggesting a predominant role of paracrine/juxtacrine factors from an endothelial origin in VM development. Via immunocytochemistry, qPCR, and secretome analyses, we determined an increased expression of proangiogenic factors as well as stemness markers in VM-forming cancer cells. Similarly, endothelial cells primed by TNBC cells showed an upregulation of proangiogenic molecules, including FGF, VEGFA, and several inflammatory cytokines. Endothelium-dependent TNBC-VM formation was prevented by AZD4547 or LY294002, strongly suggesting the involvement of the FGFR/PI3K/Akt axis in this process. Given that VM is associated with poor clinical prognosis, targeting FGFR/PI3K/Akt pharmacologically may hold promise for treating and preventing VM in TNBC tumors.

Novel Metal Nanomaterials to Promote Angiogenesis in Tissue Regeneration.

Angiogenesis-the formation of new blood vessels from existing blood vessels-has drawn significant attention in medical research. New techniques have been developed to control proangiogenic factors to obtain desired effects. Two important research areas are 1) understanding cellular mechanisms and signaling pathways involved in angiogenesis and 2) discovering new biomaterials and nanomaterials with proangiogenic effects. This paper reviews recent developments in controlling angiogenesis in the context of regenerative medicine and wound healing. We focus on novel proangiogenic materials that will advance the field of regenerative medicine. Specifically, we mainly focus on metal nanomaterials. We also discuss novel technologies developed to carry these proangiogenic inorganic molecules efficiently to target sites. We offer a comprehensive overview by combining existing knowledge regarding metal nanomaterials with novel developments that are still being refined to identify new nanomaterials.

Targeting of Notch, IL-1, and leptin has therapeutic potential in xenograft colorectal cancer.

Colorectal cancer (CRC) is a fatal malignancy type and its occurence still needs to be explored mechanistically. Notch, IL-1, and leptin crosstalk is reported to play a role in the proliferation, migration, and expression of proangiogenic molecules. In this study, we aimed to investigate the effect of inhibition of Notch, IL-1, and leptin on CRC. To generate colorectal cancer tumor xenografts, 1 × 107 cells from exponentially growing cultures of HCT-15 cells were injected subcutaneously, at the axillary region of the left and right rear flanks of forty NOD.CB17-Prkdcscid/J (NOD/SCID) female mice. The mice were then monitored for the development of tumors and were randomly divided into five groups when tumor sizes reached a volume of approximately 150 mm3. Mice were used to determine the effectiveness of the gamma-secretase inhibitor (DAPT, Notch inhibitor), the interleukin-1 receptor antagonist (Anakinra) and the leptin receptor antagonist (Allo aca) against tumor growth. The mice were euthanized by CO2 inhalation immediately after the treatments finished, and all efforts were made to minimize suffering. Tumors were excissed for RT-PCR and histological analysis. There is an intact Notch, IL-1, and leptin signaling axis, and in vivo antagonism of Notch, IL-1, and leptin affects mRNA and protein expression of inflammatory and angiogenic molecules. Present data suggest that targeting Notch, IL-1, and leptin may be possesses therapeutic potential in CRC.

Detection, Purification, Characterization, and Manipulation of Migrasomes.

Migrasomes are newly discovered cellular organelles, first described in 2015, that are formed by migrating cells. During migration, cells leave behind long membrane tethers called retraction fibers. Numerous micrometer-scale vesicles grow from the tips or junctions of these fibers, which we have named migrasomes. Migrasomes play important roles in various physiological processes, including releasing signaling factors to ensure organ morphogenesis during zebrafish embryo development, transferring mRNAs among cells, disposing of damaged mitochondria from the cell to maintain cell homeostasis, and secreting pro-angiogenic molecules to promote angiogenesis during chicken embryo development. Migrasomes are beginning to attract the attention of researchers in multiple fields. Here, we summarize the most commonly used protocols for migrasome detection using fluorescence microscopy imaging, purification through density-gradient centrifugation, characterization using electron microscopy (EM) imaging and biochemical analysis, and manipulation of migrasomes by targeting integrins and tetraspanins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Detection and observation of migrasomes by fluorescence microscopy imaging Basic Protocol 2: Purification of migrasomes from cultured cell lines and embryos by density-gradient centrifugation Basic Protocol 3: Characterization of migrasomes by electron microscopy imaging and biochemical analysis.

The interactions of docetaxel with tumor microenvironment.

There are several interactions within the tumor microenvironment (TME) that affect the response of cancer cells to therapy. There are also a large number of cells and secretions in TME that increase resistance to therapy. Following the release of immunosuppressive, pro-angiogenic, and metastatic molecules by certain cells such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and cancer cells, immune evasion, angiogenesis, and metastasis may be induced. However, natural killer (NK) cells and cytotoxic CD8+T lymphocytes (CTLs) can responsively release anticancer molecules. In addition, anticancer drugs can modulate these cells and their interactions in favor of either cancer resistance or therapy. Docetaxel belongs to taxanes, a class of anti-tumor drugs, which acts through the polymerization of tubulin and the induction of cell cycle arrest. Also, it has been revealed that taxanes including docetaxel affect cancer cells and the other cells within TME through some other mechanisms such as modulation of immune system responses, angiogenesis, and metastasis. In this paper, we explain the basic mechanisms of docetaxel interactions with malignant cells. Besides, we review the diverse effects of docetaxel on TME and cancer cells in consequence. Lastly, the modulatory effects of docetaxel alone or in conjunction with other anticancer agents on anti-tumor immunity, cancer cell resistance, angiogenesis, and metastasis will be discussed.

Adipose mesenchymal stem cell-derived soluble factors, produced under hypoxic condition, efficiently support in vivo angiogenesis

Tissue regeneration or healing both require efficient vascularization within a tissue-damaged area. Based on this concept, a remarkable number of strategies, aimed at developing new tools to support re-vascularization of damaged tissue have emerged. Among the strategies proposed, the use of pro-angiogenic soluble factors, as a cell-free tool, appears as a promising approach, able to overcome the issues concerning the direct use of cells for regenerative medicine therapy. Here, we compared the effectiveness of adipose mesenchymal stem cells (ASCs), use as cell suspension, ASC protein extract or ASC-conditioned-medium (i.e., soluble factors), combined with collagenic scaffold, in supporting in vivo angiogenesis. We also tested the capability of hypoxia in increasing the efficiency of ASC to promote angiogenesis, via soluble factors, both in vivo and in vitro. In vivo studies were performed using the Integra® Flowable Wound Matrix, and the Ultimatrix in sponge assay. Flow cytometry was used to characterize the scaffold- and sponge-infiltrating cells. Real-time PCR was used to evaluate the expression of pro-angiogenic factors by stimulating Human Umbilical-Vein Endothelial Cells with ASC-conditioned media, obtained in hypoxic and normoxic conditions. We found that, in vivo, ACS-conditioned media can support angiogenesis similar to ASCs and ASC protein extract. Also, we observed that hypoxia increases the pro-angiogenic activities of ASC-conditioned media, compared to normoxia, by generating a secretome enriched in pro-angiogenic soluble factors, with bFGF, Adiponectine, ENA78, GRO, GRO-a, and ICAM1-3, as most regulated factors. Finally, ASC-conditioned media, produced in hypoxic condition, induce the expression of pro-angiogenic molecules in HUVECs. Our results provide evidence that ASC-conditioned-medium can be proposed as a cell-free preparation able to support angiogenesis, thus providing a relevant tool to overcome the issues and restrictions associated with the use of cells.

Endocan Knockdown Down-Regulates the Expression of Angiogenesis-Associated Genes in Il-1ß Activated Chondrocytes.

Endocan is a small soluble proteoglycan (PG) known to be involved in inflammation and angiogenesis. Increased endocan expression was found in the synovia of arthritic patients and chondrocytes stimulated with IL-1ß. Considering these findings, we aimed to investigate the effects of endocan knockdown on the modulation of pro-angiogenic molecules expression in a model of IL-1ß-induced inflammation in human articular chondrocytes. Endocan, VEGF-A, MMP-9, MMP-13, and VEGFR-2 expression was measured in both normal and endocan knockdown chondrocytes stimulated with IL-1ß. VEGFR-2 and NF-kB activation were also measured. Results have shown that endocan, VEGF-A, VEGFR-2, MMP-9, and MMP-13 were significantly up-regulated during IL-1ß-induced inflammation; interestingly, the expression of such pro-angiogenic molecules and NF-kB activation were significantly reduced by endocan knockdown. These data support the hypothesis that endocan released by activated chondrocytes may be involved in the mechanisms that stimulate cell migration and invasion, as well as angiogenesis, in the pannus of arthritic joints.