In Vivo Matrigel Plug Assay as a Potent Method to Investigate Specific Individual Contribution of Angiogenesis to Blood Flow Recovery in Mice

Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for interferon regulatory factor (IRF)3, IRF7, RadioProtective 105 (RP105), Chemokine CC receptor CCR7, and p300/CBP-associated factor (PCAF) underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of IRF3, IRF7, and RP105, and a decreased angiogenesis in PCAF deficient mice. Our results also suggest an involvement of CCR7 in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.

Using of Animal Models of Angiogenesis to Confirm a Bidens pilosa-Sourced Polyacetylenic Glucoside Inhibits Angiogenesis Targeting Hypoxia through VEGF and PDIA4 Suppression

Simply Summary: Translation of new cancer treatments between pets and human were noticed in comparative oncological investigation. The current study aims at evaluating a polyacetylenic glucoside purified from an edible herb, Bidens pilosa, to present its anti-angiogenic effects. We innovatively find this polyacetylenic glucoside, cytopiloyne, shows anti-angiogenic effect on different in vitro assays and various in vivo animal models under hypoxia. Based on results of this study, cytopiloyne will be a prospective herb angiogenesis inhibitor candidate to control animal or human cancer formation as adjuvant therapy. Abstract: Anti-angiogenesis is a pivotal combination treatment approach in cancer therapy but rare using on companion animals. This study aimed at evaluating the anti-angiogenic effect of a B. pilosa sourced polyacetylenic glucoside, cytopiloyne, on various in vitro assays and in vivo models. We provide evidences showing that CP has anti-angiogenic activities. Firstly, CP inhibited sponge/ Matrigel plug angiogenesis from tumor cells and decreased the survival of tumor cells on hypoxic conditions. Besides, CP declined PKCα protein expression which a protein leads to the growth and spread of tumors under hypoxia. Secondly, inhibitory effects of CP on endothelial angiogenesis were confirmed by chick chorioallantoic membrane assay, tube formation of SVEC4-10 cells and Matrigel plug assay. A dose-dependent CP treatment inhibited 4T1 cells proliferation under hypoxia and migration. It also suppresses VEGF transcription under hypoxia. Finally, we found that CP decreased PDIA4, a novel regulator of cancer growth, expression in endothelial cells. This effect was confirmed by PDIA4 knockout mice with reduced angiogenesis in Matrigel plug assay. Taken together, these results suggest that CP might act as a promising anti-angiogenic herbal agent candidate to be used in animal hypervascularized cancer of veterinary medicine or in combination to control human cancer as adjuvant therapy.

Reservoir of Fibroblasts Promotes Recovery From Limb Ischemia

Background: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. Methods: We utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia. Results: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP + CD144 + CD11b − fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP + CD11b - cells from the plug revealed a small subset of YFP + CD144 + CD11b − E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. Conclusions: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.

bFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.

MEK / ERK inhibitor effectively impact generalized lymphatic anomaly (GLA) cells growth through EGFR / MEK /ERK signaling pathway

Background Generalized lymphatic anomaly is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation. Studies on GLA are frequently hampered by a lack of appropriate models to test the effects of potential treatments or decipher the mechanism of pathology. Moreover, diverse phenotypes observed with GLA require a large number of samples to be analyzed to obtain statistically informative results. Due to the very limited experimental material, most of the research is restricted to single case report. Methods We first time used two-step endothelial cell isolation technique (step 1: single cells were first sorted with a-human CD31 magnetic beads; step 2: collected CD31 Pos cells from step1 were sorted with a-human PDPN magnetic beads) to generate two GLA-LEC cell lines, and purified normal-LEC from normal liver tissue in the same case. To characterize the aberrant phenotype of generalized lymphatic anomaly lymphatic endothelial cells (GLA-LEC#1, and GLA-LEC#2). We investigated GLA-LECs growth curve, cell cycle, apoptosis, and sprouting angiogenesis in vitro . Matrigel plug assay was applied in immunodeficient mice to monitor the GLA-LECs formed vasculature in vivo . Rapamycin and dual MEK / ERK inhibitor were tested to investigate the efficacy on inhibiting GLA-LEC proliferation and downstream signaling pathway. Results We have successfully purified GLA-LECs from GLA tissues with > 99% purity. These cells also expressed the lymphatic markers lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and podoplanin (PDPN). GLA-LECs showed significantly higher proliferation rate compared to normal-LECs in both cases. Cell cycle analysis of cell distribution suggested that compared with normal-LECs, GLA-LECs showed increased proportion of cells in S phase and less G0/G1 phase. When GLA-LECs and normal-LECs apoptosis induced by serum deprivation, more Annexin V positive population of endothelial cells were observed in normal-LECs but not GLA-LECs. Hyper-activated epidermal growth-factor receptor (EGFR) signaling was observed in both cases of GLA-LECs, endogenously highly expression of EGF receptor and EGF induced phosphorylation of EGFR (phosphor Y1068) were found in both GLA cell lines. GLA-LECs are sensitive to both rapamycin and MEK / ERK dual inhibitor treatment. In vivo, by using Matrigel plug assay, we found both GLA-LECs and immortalized GLA-LEC (SV40) grew robust vessel-like structure. Conclusions In vitro , both GLA-LECs cell lines are highly proliferative as compared with normal-LECs. Rapamycin and dual MEK / ERK inhibitor dose-dependently inhibited GLA-LECs proliferation. In vivo , GLA-LECs showed angiogenic phenotype, and grew robust vessel-like structure in immunodeficient mice.

IN VITRO AND IN VIVO ANTIANGIOGENIC EFFECT OF ARTOCARPUS HETEROPHYLLUS SEED EXTRACT

Objective: Angiogenesis the formation of new blood vessels from the pre-existing vasculature plays a major role in tumor growth, invasion, and metastasis of cancer diseases. The current research was designed for the inhibition of angiogenesis, which can provide a novel way to inhibit tumor growth and metastasis in cancer.Methods: The antiangiogenic properties of serial concentrations of the hydroethanolic extract of Artocarpus heterophyllus were examined in human umbilical vein endothelial cells (HUVECs) using a tube formation assay in vitro and in a Matrigel plug assay as in vivo model.Results: Hydroethanolic extract of A. heterophyllus significantly inhibited vascular endothelial growth factor (VEGF)-mediated angiogenesis in the HUVECs in culture dose-dependently. Further, the new blood vessel formation was observed to be inhibited by the extract at 100 mg/kg p.o. in Matrigel plug model in C57BL/6 mice. However, the effect was enhanced in higher concentration (500 mg/kg p.o.) demonstrating the in vivo antiangiogenic activity of the extract.Conclusion: This study demonstrated that the hydroethanolic seed extract of A. heterophyllus strongly inhibited the angiogenesis in HUVECs. Moreover, the extract significantly inhibited the VEGF production in HUVECs, confirming their possible antiangiogenic mechanism.

Angiogenic Capacity of Dental Pulp Stem Cell Regulated by SDF-1α-CXCR4 Axis

Previously, the perivascular characteristics of dental pulp stem cells (DPSCs) were reported, which suggested the potential application of DPSCs as perivascular cell source. In this study, we investigated whether DPSCs had angiogenic capacity by coinjection with human umbilical vein endothelial cells (HUVECs) in vivo; in addition, we determined the role of stromal cell-derived factor 1-α(SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) axis in the mutual interaction between DPSCs and HUVECs. Primarily isolated DPSCs showed mesenchymal stem cell- (MSC-) like characteristics. Moreover, DPSCs expressed perivascular markers such as NG2,α-smooth muscle actin (α-SMA), platelet-derived growth factor receptorβ(PDGFRβ), and CD146. In vivo angiogenic capacity of DPSCs was demonstrated by in vivo Matrigel plug assay. We could observe microvessel-like structures in the coinjection of DPSCs and HUVECs at 7 days postinjection. To block SDF-1αand CXCR4 axis between DPSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into Matrigel plug. No significant microvessel-like structures were observed at 7 days postinjection. In conclusion, DPSCs have perivascular characteristics that contribute to in vivo angiogenesis. The findings of this study have potential applications in neovascularization of engineered tissues and vascular diseases.

Chemerin Produced By Mesenchymal Stromal Cells (MSC) Is an Important Factor for In Vivo macrophage Migration

Mesenchymal Stromal Cells (MSC) are multipotent cells currently used for treating several inflammatory disorders thanks to their ability to modulate the immune response. However, the mechanisms by which MSC are able to suppress the immune response have not been fully understood. Chemerin has been recently identified as a chemotactic protein, secreted as a precursor, named Prochemerin, and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain by different serine and cysteine proteases derived from the fibrinolitic, coagulation and inflammatory cascade. In particular, we observed that both human and mouse bone marrow-derived MSC were able to produce Chemerin under basal conditions and its production was strongly increased after stimulation with inflammatory cytokines. The aim of this study was to understand whether Chemerin produced by MSC is involved in their potent immune-modulatory activity. Chemerin was immune-purified from supernatant of human MSC (MSC-Chem) and utilized for measuring in vitro migration index (MI) of pre-B cells expressing the human ChemR23 receptor (L1.2-ChemR23). MSC-Chem was able to induce the migration of ChemR23-expressing cells in a dose-depend manner (MI 1nM=85, MI 5nM=480, MI 10nM=1131). However, recombinant human (rh)-chemerin induced higher migration of L1.2-ChemR23 cells compared to MSC-Chem (MI 5nM=1938), suggesting that only a fraction of MSC-Chem was converted into its active form by MSC themselves. In accordance, LC/MS mass spectrometry analysis on purified MSC-Chem did not identify the active form of the protein. Interestingly, pre-incubation of MSC-Chem with Neutrophil Elastase and Cathepsin L induced a strong migration of L1.2-ChemR23 cells compared to MSC-Chem alone (MI MSC-Chem alone 1 nM=23.33; MI MSC-Chem 1 nM + Neutrophil Elastase=328; MI MSC-Chem 1 nM + Cathepsin L=4950; p=0.002), suggesting that MSC-Chem were converted in its active form, after cleavage by proteases. Starting from these data, we established an in vivo migration assay by injecting under the abdominal skin of C57BL6 mice a mix of matrigel and murine (m)MSC (secreting or not Chemerin). After 5 days, the matrigel plug was excided, digested and infiltrating immune cells were analyzed by FACS analysis. Chemerin production by mMSC was totally abrogated by using RNA interference approach (sChem-MSC). Interestingly, mMSC features, such as phenotype and differentiation ability, were not affected by the gene-silencing process. Preliminary results showed that 5 days after injection, scramble Chem-MSC were able to recruit macrophages (CD45+CD11b+F4/80+ cells) into the matrigel plug. On the other hand, sChem-MSC drastically decreased their ability to induce macrophages migration, (sChem-MSC mean=2.38%, range=0.8%-6.4%; scramble MSC mean=8.2%; range=4%-11.5%; p=0.01; n=3). These findings identify a new mechanism by which MSC, through Chemerin production, attract macrophages in vivo. Further studies are needed to understand whether recruited macrophages are also affected by the immunomodulatory activity of MSC Disclosures No relevant conflicts of interest to declare.

Inhibitory Effect of Endostar on Specific Angiogenesis Induced by Human Hepatocellular Carcinoma

To investigate the effect of endostar on specific angiogenesis induced by human hepatocellular carcinoma, this research systematically elucidated the inhibitory effect on HepG2-induced angiogenesis by endostar from 50 ng/mL to 50000 ng/mL. We employed fluorescence quantitative Boyden chamber analysis, wound-healing assay, flow cytometry examination using a coculture system, quantitative analysis of tube formation, andin vivoMatrigel plug assay induced by HCC conditioned media (HCM) and HepG2 compared with normal hepatocyte conditioned media (NCM) and L02. Then, we found that endostar as a tumor angiogenesis inhibitor could potently inhibit human umbilical vein endothelial cell (HUVEC) migration in response to HCM after four- to six-hour action, inhibit HCM-induced HUVEC migration to the lesion part in a dose-dependent manner between 50 ng/mL and 5000 ng/mL at 24 hours, and reduce HUVEC proliferation in a dose-dependent fashion. Endostar inhibited HepG2-induced tube formation of HUVECs which peaked at 50 ng/mL.In vivoMatrigel plug formation was also significantly reduced by endostar in HepG2 inducing system rather than in L02 inducing system. It could be concluded that, at cell level, endostar inhibited the angiogenesis-related biological behaviors of HUVEC in response to HCC, including migration, adhesion proliferation, and tube formation. At animal level, endostar inhibited the angiogenesis in response to HCC in Matrigel matrix.