Vessel Maturation Research Articles

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

Development of a bioreactor system for pre-endothelialized cardiac patch generation with enhanced viscoelastic properties by combined collagen I compression and stromal cell culture.

Treatment of terminal heart failure still poses a significant clinical problem. Cardiac tissue engineering could offer autologous solutions for the replacement of nonfunctional myocardial tissue. So far, soft matrix construction and missing large-scale prevascularization prevented the application of sizeable cardiac repair patches. We developed a novel bioreactor system for semi-automatic compression of a collagen I hydrogel applying 16 times higher pressure than in previous studies. Resistance towards compression stress was investigated for multiple cardiac-related cell types. For scaffold prevascuarization, a tubular cavity was imprinted during the compaction process. Primary cardiac-derived endothelial cells (ECs) were isolated from human left atrial appendages (HLAAs) and characterized by fluorescence-activated cell sorting (FACS) and immunocytology. EC were then seeded into the preformed channel with dermal fibroblasts as interstitial cell component of the fully cellularized patch. After 8 days of constant perfusion culture within the same bioreactor, scaffold dynamic modulus and cell viability were analyzed. Endothelial proliferation and vessel maturation were examined by immunohistochemistry and transmission electron microscopy. Our design allowed for scaffold production and dynamic culture in a one-stop-shop model. Enhanced compression and cell-mediated matrix remodeling induced a significant increase in scaffold stiffness while ensuring excellent cell survival. For the first time, we could isolate HLAA-derived EC with proliferative potential. ECs within the central channel proliferated during flow culture, continuously expressing endothelial markers (CD31) and displaying basal membrane synthesis (collagen IV, ultrastructural analysis). After 7 days of culture, a complete endothelial monolayer could be observed. Covering cells aligned themselves in flow direction and developed mature cell-cell contacts.

Multipotent adult progenitor cells grown under xenobiotic-free conditions support vascularization during wound healing

BackgroundCell therapy has been evaluated pre-clinically and clinically as a means to improve wound vascularization and healing. While translation of this approach to clinical practice ideally requires the availability of clinical grade xenobiotic-free cell preparations, studies proving the pre-clinical efficacy of the latter are mostly lacking. Here, the potential of xenobiotic-free human multipotent adult progenitor cell (XF-hMAPC®) preparations to promote vascularization was evaluated.MethodsThe potential of XF-hMAPC cells to support blood vessel formation was first scored in an in vivo Matrigel assay in mice. Next, a dose-response study was performed with XF-hMAPC cells in which they were tested for their ability to support vascularization and (epi) dermal healing in a physiologically relevant splinted wound mouse model.ResultsXF-hMAPC cells supported blood vessel formation in Matrigel by promoting the formation of mature (smooth muscle cell-coated) vessels. Furthermore, XF-hMAPC cells dose-dependently improved wound vascularization associated with increasing wound closure and re-epithelialization, granulation tissue formation, and dermal collagen organization.ConclusionsHere, we demonstrated that the administration of clinical-grade XF-hMAPC cells in mice represents an effective approach for improving wound vascularization and healing that is readily applicable for translation in humans.

Shaping Waves of Bone Morphogenetic Protein Inhibition During Vascular Growth.

The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

A metabolite roadmap of the wood-forming tissue in Populus tremula.

Wood, or secondary xylem, is the product of xylogenesis, a developmental process that begins with the proliferation of cambial derivatives and ends with mature xylem fibers and vessels with lignified secondary cell walls. Fully mature xylem has undergone a series of cellular processes, including cell division, cell expansion, secondary wall formation, lignification and programmed cell death. A complex network of interactions between transcriptional regulators and signal transduction pathways controls wood formation. However, the role of metabolites during this developmental process has not been comprehensively characterized. To evaluate the role of metabolites during wood formation, we performed a high spatial resolution metabolomics study of the wood-forming zone of Populus tremula, including laser dissected aspen ray and fiber cells. We show that metabolites show specific patterns within the wood-forming zone, following the differentiation process from cell division to cell death. The data from profiled laser dissected aspen ray and fiber cells suggests that these two cell types host distinctly different metabolic processes. Furthermore, by integrating previously published transcriptomic and proteomic profiles generated from the same trees, we provide an integrative picture of molecular processes, for example, deamination of phenylalanine during lignification is of critical importance for nitrogen metabolism during wood formation.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

The long-term effects of anti-vascular endothelial growth factor therapy on the optical coherence tomography angiographic appearance of neovascularization in age-related macular degeneration

BackgroundThe short-term effects of anti-vascular endothelial growth factor (anti-VEGF) treatment on macular neovascularization (MNV) morphology is well described, but long-term studies on morphologic changes and correlation of such changes to the type of MNV have not been conducted. This study aims to determine if different types of MNVs in neovascular AMD (nAMD) behave differently with anti-VEGF treatment as visualized on optical coherence tomography angiography (OCTA).MethodsTreatment-naïve nAMD patients were retrospectively screened for baseline and follow-up OCTA imaging 10 or more months after initial treatment. Images were graded for MNV type, area, activity, mature versus immature vessels, vessel density, presence of atrophy, atrophy location and area. Growth rate was calculated as the percent change in lesion area from baseline over the years of follow-up. In addition, the occurrence of complete regression and the percent of lesions that grew, remained stable, and shrunk per type was also evaluated.ResultsForty-three eyes from 43 patients with a mean follow-up of 2 years were evaluated. On structural OCT, 26 lesions were classified as pure type 1 MNVs, 12 MNVs had a type 2 component, and 5 MNVs had a type 3 component. Of these cases, 2 mixed-type MNVs were considered to have completely regressed. There was no significant differences in MNV area and growth rate between type 1 and type 2 lesions, but all cases of type 3 lesions shrunk in the follow-up period. There was no correlation between the number of injections per year and growth rate, endpoint MNV area or endpoint activity status for any MNV type. There was no significant association between the development of atrophy and the number of injections, baseline MNV area, baseline vessel density, or lesion growth rate.ConclusionsIn nAMD, complete regression of an MNV network exposed to anti-VEGF is rare. This work emphasizes the role of anti-VEGF as anti-leakage rather than vascular regression agents in nAMD.

Follicle populations and vascularization in ovarian tissue of pediatric patients before and after long-term grafting

To characterize ovarian tissue from pediatric patients by evaluating development and vascularization in follicle populations and comparing it with adult tissue after xenografting. Prospective experimental study. Academic research center. Five children (median age 3 years) and seven women (median age 28 years). Hematoxylin and eosin staining, immunofluorescence, and transmission electron microscopy (TEM) evaluation before and after grafting. Follicle density, morphology, classification, and size, ovarian tissue vascularization, follicle ultrastructure. Frozen-thawed ovarian tissue was divided into three fragments: nongrafted controls, TEM, and xenografting for 20 weeks. Follicle density was statistically significantly higher in pediatric than adult patients; even though it decreased in both groups after transplantation, it remained higher in pediatric patients. In the pediatric group, quiescent-stage follicles were the majority of the follicle pool before and after grafting, while growing follicles statistically significantly increased in both groups after grafting. Abnormal and atretic follicles were also observed in pediatric tissue and declined with age and after grafting. Pediatric ovarian tissue contained more and larger immature vessels, while mature vessels were larger in adults. The TEM analysis of abnormal pediatric follicles showed loss of shape and vacuolization of the cytoplasm without organelle damage. Statistically significant differences in follicle density were observed between pediatric and adult patients, but the follicle proportions were similar before and after grafting, with the exception of atretic and abnormal follicles. Pediatric tissue contains more and larger immature vessels than adult tissue, and the posttransplantation revascularization process is accelerated in this group.

Application of IVIM-DWI in Detecting the Tumor Vasculogenic Mimicry Under Antiangiogenesis Combined With Oxaliplatin Treatment.

Objectives: This study aimed to detect the time window of vascular normalization during anti-vascular treatment using intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI). Simultaneously, we evaluated the tumor invasiveness and vasculogenic mimicry and performed synthetic assessment of treatment efficacy of angiogenesis inhibitor combined with conventional chemotherapy using IVIM-DWI.Materials and Methods: HCT116 cells were subcutaneously administered into the right flank of BALB/C nude mice to build a colon cancer xenograft model. Thirty-two tumor-bearing mice were randomly divided into four groups and intraperitoneally administered with normal saline (Group A or control group), bevacizumab (Group B), oxaliplatin monotherapy (Group C), and oxaliplatin combined with bevacizumab (Group D). The IVIM-DWI was performed on days 0, 3, 6, 9, 12, and 15 after the treatments. Another 51 tumor-bearing mice were included in the pathological examinations. α-Smooth muscle actin (SMA) and CD31 double-staining, periodic acid-Schiff (PAS) and CD31 double-staining, hematoxylin and eosin (HE), Ki-67, and E-cadherin staining were performed. The tumor growth and dynamic change of each parameter were noted.Results: The mice in Group D manifested the smallest tumor volume and highest tumor inhibition rate. Microvessel density was significantly decreased but accompanied by increased vasculogenic mimicry after antiangiogenic treatment. The trend was reversed by oxaliplatin treatment. Treated with bevacizumab, the vessel maturity index shared a similar trend with D* and f-values during days 3–12, which slowly increased from days 0 to 9 and then decreased briefly. D-value significantly correlated with vasculogenic mimicry and Ki-67, while D* and f-values showed positive correlations with microvessel density and E-cadherin, an indicator of epithelial–mesenchymal transition.Conclusion: Oxaliplatin performed an inhibited effect on vasculogenic mimicry. Bevacizumab can enhance the tumor chemotherapy through vascular normalization within a transient time period, which can be detected by IVIM-DWI. D* and f-values are able to predict the tumor invasiveness while D is superior in reflecting vasculogenic mimicry and Ki-67 expression during antitumor treatment.

MTOR Promotes Tissue Factor Expression and Activity in EGFR-Mutant Cancer.

Mechanistic target of rapamycin (mTOR) signaling pathway mediates the function of oncogenic receptor tyrosine kinases (RTKs). We aimed to elucidate new role of mTOR in EGFR-mutant (EGFR-mut) non-small cell lung cancer (NSCLC) and glioblastoma (GBM) with a focus on tumor microenvironments. Here, we report a novel regulatory link between mTOR complexes (mTORCs) and tissue factor (TF), an initiator of tumor-derived thrombosis. TF is elevated in EGFR-mut NSCLC/GBM cell lines and tumors from patients with poor prognosis. Application of mTORC1/2 inhibitors (AZD8055, WYE-125132, MTI-31, and rapamycin) or genetic mTORC-depletion all reduced TF expression, which appeared to be differentially mediated depending on cellular context. In U87MG and HCC827 cells, mTORC1 exerted a dominant role via promoting TF mRNA transcription. In EGFR-TKI-resistant H1975 and PC9 cells, it was mTORC2 that played a major role in specific repression of lysosomal-targeted TF protein degradation. Successful inhibition of TF expression was demonstrated in AZD8055- or MTI-31-treated H1975 and U87MG tumors in mice, while a TF-targeted antibody antagonized TF activity without reducing TF protein. Both the mTOR- and TF-targeted therapy induced a multifaceted remodeling of tumor microenvironment reflecting not only a diminished hypercoagulopathy state (fibrin level) but also a reduced stromal fibrosis (collagen distribution), compromised vessel density and/or maturity (CD31 and/or α-SMA) as well as a substantially decreased infiltration of immune-suppressive M2-type tumor-associated macrophages (CD206/F4/80 ratio). Thus, our results have identified TF as a functional biomarker of mTOR. Downregulation of mTOR-TF axis activity likely contributes to the therapeutic mechanism of mTORC1/2- and TF-targeted agents in EGFR-mut advanced NSCLC and GBM.

Abstract 2645: Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications

Abstract Obesity is associated with an increased risk of, and a poor prognosis for, postmenopausal (PM) breast cancer (BC). The goal of this study was to determine whether diet-induced obesity (DIO) promotes (i) shorter tumor latency, (ii) escape from tumor dormancy, and (iii) acceleration of tumor growth, angiogenesis and metastasis and to elucidate the underlying mechanism/s. Two different BC models, an orthotopic model (luciferase/MDA-MB-436 injected into the mammary fat pad (MFP) of SCID mice) and a transgenic model (doxycycline-driven conditional MMTV/TWNT/luciferase) were utilized. To model the effect of obesity on PM BC, mice were ovariectomized (OVX) before being placed on a DIO (obese) or normal chow (NC; lean) regimen. In both the orthotopic and transgenic BC models, obese mice had significantly higher tumor frequency, higher tumor volume, and lower overall survival compared to lean mice. Breast tumors in OVX/DIO mice had significantly higher microvessel density (MVD), SMA+ mature vessels and increased aggressive local invasion into the surrounding fat pad and muscle. OVX/DIO TWNT mice displayed a significantly shorter tumor latency period (7 vs. >24 wk, P=0.003) compared to OVX/NC mice. Levels of VEGF, IL-6, bFGF, Lcn-2 and MMP-9 were higher, whereas Tsp-1 levels were lower, in the sera/urine of DIO mice during tumor progression, suggesting that these circulating factors could serve as surrogate biomarkers of BC development. OVX/DIO tumors had increased expression of the angiogenic factors VEGF and bFGF and higher pVEGFR2. Systematic time-course analysis of MFP in the TWNT BC model indicated that DIO MFP achieved advanced tumor proliferative and angiogenic status at significantly earlier time points compared to NC MFP. DIO MFP also exhibited an increased incidence of extensive hyperplasia and carcinoma in situ at earlier time points, suggesting that obesity promotes tumor initiation as well as progression. Hyperplastic ducts and microscopic neoplastic lesions (≤1mm) in DIO MFP in the early phases of tumor development displayed increased Ki67 staining and were associated with significantly higher numbers of blood vessels. Collectively, our data suggest that PM obesity reduces breast tumor latency, promotes escape from tumor dormancy and initiates aggressive tumor growth via increased angiogenesis. Consistent with these findings, targeting angiogenesis via a small molecule inhibitor resulted in delayed tumor formation compared to vehicle-treated mice in the OVX/DIO TWNT BC model, suggesting that targeting angiogenesis may be a useful strategy in obesity-driven breast tumor initiation and progression. Ongoing studies in our laboratory are investigating the mechanism(s) by which an obesogenic microenvironment mediates these effects. [This work was supported by NIH RO1CA185530, the Karp Family Foundation and the Breast Cancer Research Foundation] Citation Format: Roopali Roy, Jiang Yang, Lauren Merritt, Justine Alluine, Lewis A. Chodosh, Marsha A. Moses. Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2645.

Abstract 1477: LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy

Abstract In cancer blood vessels are dysfunctional, poorly perfused and leaky. Malfunctioning vessels contribute to the pro-oncogenic environment and limit the efficacy of current systemically administered drugs. Normalizing the tumor vasculature to improve vessel permeability, reduce hypoxia and vascular leakage and enhance drug delivery, has become an experimental objective in cancer research. This study was carried out to investigate the effect of blocking the secreted glycoprotein leucine-rich alpha-2-glycoprotein 1 (LRG1) on tumor vascular function, and evaluate the impact it has on the efficacy of the common standard of care chemotherapeutic drug cisplatin. Under normal conditions LRG1 is mainly expressed in the liver but also in other tissues such as bone marrow and immune cells. LRG1 has been described in multiple reports to be a serum prognostic biomarker in several cancers, for example lung, prostate, colorectal and breast. LRG1 promotes dysfunctional vessel growth by disrupting TGFβ signaling. We demonstrate that in Lrg1-/- mice and following treatment with a LRG1 function-blocking antibody (15C4) tumor growth was inhibited. In addition, we show using RNAscope that following subcutaneous grafting of the B16F0 and LL2 tumor cell lines in mice, Lrg1 is induced in tumor endothelial cells. Despite having no effect on total vessel area, the density was decreased upon LRG1 blockade, with the persisting larger vessels exhibiting improved vessel structure as evidenced by increased pericyte and basement membrane endothelial cell coverage. Better mural cell association with tumor vascular endothelial cells and basement membrane coverage are also indicators of vessel stabilization and maturation. Using a systemically delivered fluorescent lectin tracer to mark perfused vessels, we observed a significant increase in tumor perfusion in mice treated with 15C4. Lastly, vessel normalization, through LRG1 antibody blockade, significantly enhanced the efficacy of cisplatin chemotherapy as shown by a slower tumor growth rate and increased tumor cell death compared to monotherapy. These data further corroborate the hypothesis that inhibition of LRG1 improves the delivery, and hence efficacy, of a cytotoxic drug. In conclusion, deletion or inhibition of LRG1 results in an improved vascular configuration and function, and the efficacy of chemotherapy. LRG1 blockade may therefore represent a novel strategy to enhance vessel health and improve the efficacy of cancer therapeutics. Citation Format: Camilla Pilotti, Marie N O'Connor, David Kallenberg, Laura Dowsett, Jestin George, Stephen E. Moss, John Greenwood. LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1477.

Abstract 1483: A novel anti-Tie2 antibody stabilizes vessel and increases tumor infiltrated lymphocytes

Abstract Objective: The delivery of drugs and immune cells into the tumor microenvironment is limited due to abnormal tumor vessel which is characterized as low oxygen concentration, low perfusion rate and leakage. It leads to diminish the efficacy of drugs and radiation therapy. Traditional anti-angiogenesis strategies attempt to reduce the tumor vascular supply, but their success is restricted by insufficient efficacy or development of resistance. To solve the problem, we try to using active vessel stabilizer, which can normalize the abnormal condition of tumor vessel. Background: Tie2 acts as cell-surface receptor for Ang1/Ang2 and regulates angiogenesis, endothelial cell survival, proliferation, migration, as well as maintenance of vascular quiescence. Especially, Ang1-Tie2 signaling pathway has been known to promote blood vessel maturation and stabilization. Methods: We have developed a series of novel human IgG monoclonal antibody that binds to human-TIE2 with a high affinity (Kd ~0.1 nM). The antibody binds to human TIE2 extracellular domain and activates its associated function in a ligand independent way. The mode of action was studied in vitro with human umbilical vein endothelial cells (HUVEC) and animal models. The vessel stabilization was investigated with laser induced choroidal neovascularization (CNV). The effect on tumor infiltrated lymphocyte was also studied in mouse colon cancer model. Results: Anti-Tie2 antibody increased human endothelial cell migration like Ang1 and inhibited VEGF-induced vascular leakage, contributing to vessel normalization. It binds to Tie2 in a ligand independent way and thus it can stabilize vessel as an agonist. Both p-VEGFR and p-VE-Cadherin levels are also significantly decreased on VEGF-induced VEGFR signaling pathway by anti-Tie2 antibody, suggesting that it can maintain cell to cell junction. The vessel stabilization effect was demonstrated in a mouse model of laser induced CNV. Moreover, in syngeneic CT26 mouse models, anti-Tie2 antibody showed anti-tumor effect in which it significantly reduced the tumor growth and synergistically when it combined with anti PD-1 antibody. The tumor infiltrating lymphocytes were increased in CT26 tumor, especially CD8+ T cells and MDSC when anti-Tie2 was administered. Conclusion: We developed active vessels stabilizer targeting Tie2, having an effect of vessel stabilization in a ligand independent way. It inhibited VEGF-induced vascular leakage through decrease of p-VEGFR and VE-cadherin. This active vessel stabilizer could help deliver drug and lymphocyte into the TME. The therapeutic efficacy of immune checkpoint inhibitors can be increased when it combined with this active vessel stabilizer. Thus, this active vessel stabilizer can change cold tumor into hot tumor. Citation Format: Eun-Ah Lee, Beomyong Park, Nu Ri Kang, Youngae Lee, Do-yun Kim, Sang Soon Byun, Jin Yong An, Cheon Ho Park, Weon Sup Lee, Joo Hyoung Lee. A novel anti-Tie2 antibody stabilizes vessel and increases tumor infiltrated lymphocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1483.

Oleic Acid, Cholesterol, and Linoleic Acid as Angiogenesis Initiators.

The current study determined the natural angiogenic molecules using an unbiased metabolomics approach. A chick chorioallantoic membrane (CAM) model was used to examine pro- and antiangiogenic molecules, followed by gas chromatography–mass spectrometry (GCMS) analysis. Vessel formation was analyzed quantitatively using the angiogenic index (p < 0.05). At embryonic day one, a white streak or circle area was observed when vessel formation begins. GCMS analysis and database search demonstrated that angiogenesis may initiate when oleic, cholesterol, and linoleic acids increased in the area of angiogenic reactions. The gain of function study was conducted by the injection of cholesterol and oleic acid into a chick embryo to determine the role of each lipid in angiogenesis. We propose that oleic acid, cholesterol, and linoleic acid are natural molecules that set the platform for the initiation stage of angiogenesis before other proteins including the vascular endothelial growth factor, angiopoietin, angiotensin, and erythropoietin join as the angiome in sprout extension and vessel maturation.

Dapagliflozin Promotes Neovascularization by Improving Paracrine Function of Skeletal Muscle Cells in Diabetic Hindlimb Ischemia Mice Through PHD2/HIF-1α Axis.

Diabetes mellitus is associated with a high risk of hindlimb ischemia (HLI) progression and an inevitably poor prognosis, including worse limb salvage and mortality. Skeletal muscle cells can secrete angiogenic factors, which could promote neovascularization and blood perfusion recovery. Thus, paracrine function of skeletal muscle cells, which is aberrant in diabetic conditions, is crucial for therapeutic angiogenesis in diabetic HLI. Dapagliflozin is a well-known anti-hyperglycemia and anti-obesity drug; however, its role in therapeutic angiogenesis is unknown. Herein, we found that dapagliflozin could act as an angiogenesis stimulator in diabetic HLI. We showed that dapagliflozin enhances the viability, proliferation, and migration potentials of skeletal muscle cells and promotes the secretion of multiple angiogenic factors from skeletal muscle cells, most plausibly through PHD2/HIF-1α axis. Furthermore, we demonstrated that conditioned medium from dapagliflozin-treated skeletal muscle cells enhances the proliferation and migration potentials of vascular endothelial and smooth muscle cells, which are two fundamental cells of functional mature vessels. Finally, an in vivo study demonstrated that intramuscular administration of dapagliflozin effectively enhances the formation of mature blood vessels and, subsequently, blood perfusion recovery in diabetic HLI mice. Hence, our results suggest a novel function of dapagliflozin as a potential therapeutic angiogenesis agent for diabetic HLI.

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

S1PR1 regulates the quiescence of lymphatic vessels by inhibiting laminar shear stress-dependent VEGF-C signaling.

During the growth of lymphatic vessels (lymphangiogenesis), lymphatic endothelial cells (LECs) at the growing front sprout by forming filopodia. Those tip cells are not exposed to circulating lymph, as they are not lumenized. In contrast, LECs that trail the growing front are exposed to shear stress, become quiescent, and remodel into stable vessels. The mechanisms that coordinate the opposed activities of lymphatic sprouting and maturation remain poorly understood. Here, we show that the canonical tip cell marker Delta-like 4 (DLL4) promotes sprouting lymphangiogenesis by enhancing VEGF-C/VEGF receptor 3 (VEGFR3) signaling. However, in lumenized lymphatic vessels, laminar shear stress (LSS) inhibits the expression of DLL4, as well as additional tip cell markers. Paradoxically, LSS also upregulates VEGF-C/VEGFR3 signaling in LECs, but sphingosine 1-phosphate receptor 1 (S1PR1) activity antagonizes LSS-mediated VEGF-C signaling to promote lymphatic vascular quiescence. Correspondingly, S1pr1 loss in LECs induced lymphatic vascular hypersprouting and hyperbranching, which could be rescued by reducing Vegfr3 gene dosage in vivo. In addition, S1PR1 regulates lymphatic vessel maturation by inhibiting RhoA activity to promote membrane localization of the tight junction molecule claudin-5. Our findings suggest a potentially new paradigm in which LSS induces quiescence and promotes the survival of LECs by downregulating DLL4 and enhancing VEGF-C signaling, respectively. S1PR1 dampens LSS/VEGF-C signaling, thereby preventing sprouting from quiescent lymphatic vessels. These results also highlight the distinct roles that S1PR1 and DLL4 play in LECs when compared with their known roles in the blood vasculature.

Effects of controlled dual growth factor delivery on bone regeneration following composite bone-muscle injury

The objective of this study was to investigate the controlled release of two growth factors (BMP-2 and VEGF) as a treatment strategy for bone healing in clinically challenging composite injuries, consisting of a femoral segmental bone defect and volumetric muscle loss. This is the first investigation of dual growth factor delivery in a composite injury model using an injectable delivery system consisting of heparin microparticles and alginate gel. The loading efficiency of growth factors into these biomaterials was found to be >90%, revealing a strong affinity of VEGF and BMP-2 to heparin and alginate. The system could achieve simultaneous or tunable release of VEGF and BMP-2 by varying the loading strategy. Single growth factor delivery (VEGF or BMP-2 alone) significantly enhanced vascular growth in vitro. However, no synergistic effect was observed for dual growth factor (BMP-2 + VEGF) delivery in vitro. Effective bone healing was achieved in all treatment groups (BMP-2, simultaneous or tunable delivery of BMP-2 and VEGF) in the composite injury model. The mechanics of the regenerated bone reached a maximum strength of ~52% of intact bone with tunable delivery of VEGF and BMP-2. Overall, simultaneous or tunable co-delivery of low-dose BMP-2 and VEGF failed to fully restore the mechanics of bone in this injury model. Given the severity of the composite injury, VEGF alone may not be sufficient to establish mature and stable blood vessels when compared with previous studies co-delivering BMP-2+VEGF enhanced bone tissue regeneration. Hence, future studies are warranted to develop an alternative treatment strategy focusing on better control over growth factor dose, spatiotemporal delivery, and additional growth factors to regenerate fully functional bone tissue. Statement of SignificanceWe have developed an injectable delivery system consisting of heparin microparticles and an alginate hydrogel that is capable of delivering multiple growth factors in a tunable manner. We used this delivery system to deliver BMP-2 and VEGF in a rodent model of composite bone-muscle injury that mimics clinical type III open fractures. An advanced treatment strategy is necessary for these injuries in order to avoid the negative side effects of high doses of growth factors and because it has been shown that the addition of a muscle injury in this model attenuates the bone regenerative effect of BMP-2. This is the first study to test the effects of dual growth factor delivery (BMP-2/VEGF) on bone healing in a composite bone-muscle injury model and is expected to open up new directions in protein delivery for regenerative medicine.

New Insights into Diffuse Large B-Cell Lymphoma Pathobiology

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL), accounting for about 40% of all cases of NHL. Analysis of the tumor microenvironment is an important aspect of the assessment of the progression of DLBCL. In this review article, we analyzed the role of different cellular components of the tumor microenvironment, including mast cells, macrophages, and lymphocytes, in the tumor progression of DLBCL. We examined several approaches to confront the available pieces of evidence, whereby three key points emerged. DLBCL is a disease of malignant B cells spreading and accumulating both at nodal and at extranodal sites. In patients with both nodal and extranodal lesions, the subsequent induction of a cancer-friendly environment appears pivotal. The DLBCL cell interaction with mature stromal cells and vessels confers tumor protection and inhibition of immune response while delivering nutrients and oxygen supply. Single cells may also reside and survive in protected niches in the nodal and extranodal sites as a source for residual disease and relapse. This review aims to molecularly and functionally recapitulate the DLBCL–milieu crosstalk, to relate niche and pathological angiogenic constitution and interaction factors to DLBCL progression.

Therapeutic angiogenesis with exosomal microRNAs: an effectual approach for the treatment of myocardial ischemia.

Therapeutic angiogenesis presents a potential approach for treating ischemic heart diseases especially in patients who are not appropriate candidates for traditional approaches of revascularization. This approach acts through inducing the neovascularization or maturation of pre-existing collateral vessels into functional arteries to bypass the blocked arteries and restore perfusion to ischemic myocardium. Successful stimulation of local angiogenesis can be established by the cross talk between stem cells, endothelial cells, and cardiomyocytes, which is mainly mediated by paracrine communication accompanied by secreted exosomes. Exosomes are extracellular vesicles carrying a complex of signaling molecules, such as microRNAs (miRs) that can modulate the function of recipient cells. Such particles have been indicated to exert cardioprotective role through providing signaling cues for angiogenesis, an effect ascribed mainly to their miRs content. Exosomal miRs-mediated therapeutic angiogenesis has been under drastic preclinical and clinical studies. In the current review, it was aimed to summarize pro-angiogenic exosomal miRs released by various cell types mediating angiogenesis, including stem cells, endothelial cells, and cardiomyocytes, which appear to exert a therapeutic effect on the myocardial ischemia. In brief, secreted exosomal miRs including miR-210, miR-23a-3p, miR-424, let-7f, miR-30b, miR-30c, miR-126, miR-21, miR-132, miR-130a-3p, miR-214, miR-378, miR-126, miR-133, and let-7b-5p could protect against myocardial ischemia through inducing cardiac angiogenesis and vascular regeneration resulting in the increase blood flow to ischemic myocardium.