Endothelial Migration Assays Research Articles

Evaluation of a high-throughput in vitro endothelial cell migration assay for the assessment of nicotine and tobacco delivery products

Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making.Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay.3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.

Interleukin 35 Expression Correlates With Microvessel Density in Pancreatic Ductal Adenocarcinoma, Recruits Monocytes, and Promotes Growth and Angiogenesis of Xenograft Tumors in Mice

Cells of the monocyte lineage contribute to tumor angiogenesis. Interleukin 35 (IL35) is a member of the IL12 family produced by regulatory, but not effector, T cells. IL35 is a dimer comprising the IL12 alpha and IL27 beta chains, encoded by IL12A and EBI3, respectively. Expression of IL35 is increased in pancreatic ductal adenocarcinomas (PDACs) compared with normal pancreatic tissues, and promotes metastasis. We investigated the role of IL35 in monocyte-induced angiogenesis of PDAC in mice. We measured levels of IL35 protein, microvessel density, and numbers of monocytes in 123 sequential PDAC tissues from patients who underwent surgery in China in 2010. We performed studies with the human PDAC cell lines CFPAC-1, BxPC-3, Panc-1, MIA-PaCa-2, and mouse PDAC cell line Pan02. Monocyte subsets were isolated by flow cytometry from human peripheral blood mononuclear cells. Fused human or mouse IL12A and EBI3 genes were overexpressed in PDAC cells or knocked down using small hairpin RNAs. Cells were grown as xenograft tumors in SCID mice; some mice were given injections of an IL35-neutralizing antibody and tumor growth was monitored. We performed chemotaxis assays to measure the ability of IL35 to recruit monocytes. We analyzed mRNA sequences of 179 PDACs in the Cancer Genome Atlas to identify correlations between expression of IL12A and EBI3 and monocyte markers. Monocytes incubated with IL35 or PDAC cell supernatants were analyzed in tube formation and endothelial migration assays. In PDAC samples from patients, levels of IL35 mRNA and protein correlated with microvessel density and infiltration of monocyte lineage cells. In cells and mice with xenograft tumors, IL35 increased recruitment of monocytes into PDAC tumors, which required CCL5. Upon exposure to IL35, monocytes increased expression of genes whose products promote angiogenesis (CXCL1 and CXCL8). IL35 activated transcription of CCL5, CXCL1, and CXCL8 by inducing GP130 signaling, via IL12RB2 and phosphorylation of STAT1 and STAT4. A combination of a neutralizing antibody against IL35 and gemcitabine significantly decreased monocyte infiltration, microvessel density, and volume of xenograft tumors grown from PDAC cells in mice. PDAC cells produce IL35 to recruit monocytes via CCL5 and induce macrophage to promote angiogenesis via expression of CXCL1 and CXCL8. IL35 signaling promotes angiogenesis and growth of xenograft tumors from PDAC cells in mice. IL35 might serve as a therapeutic target for patients with pancreatic cancer.

A Tobacco-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice.

Thymosin β4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tβ4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tβ4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

Mac1+/Gr1+ cells contribute to transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is a serious complication associated with blood transfusion and can cause transfusion associated fatalities. Both antibody dependent and non-dependent mechanisms are involved in TRALI, as proposed over the past years. Nonetheless, many details of the immune cells involved in TRALI, particularly the Mac1(+)/Gr1(+) cells from donors, are not fully understood yet. Here we used an in vitro transwell system and a mouse model to study the role of donor leukocytes, present in the donor material, in the occurrence of TRALI reactions. We found that there is a number of immature myeloid cells with Mac1(+)/Gr1(+) phenotype present in the red blood cell (RBC) products, when prepared by regular methods. We found that murine Mac1(+)/Gr1(+) cells from stored RBC products display an elevated MHC I and CD40 expression, as well as an enhanced tumor necrosis factor alpha(TNF-α), interlukin-6(IL-6) and macrophage inflammatory protein 2 (MIP-2) secretion. When tested in a transwell endothelial migration assay, Mac1(+)/Gr1(+) cells showed a significant capability to cross the endothelial barrier. In vivo investigation demonstrated that compared to the purified RBC transfusion, more murine Mac1(+)/Gr1(+) cells from the regular method produced RBC sequestered in the lung, which associated to shorter survival. Taken together, these data suggest that donor derived Mac1(+)/Gr1(+) cells can play a significant role in TRALI reactions, and that reduction of Mac1(+)/Gr1(+) cell number from RBC products is necessary to control the severity of TRALI reactions in clinic.

Short synthetic endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse model

To determine the active peptide regions inside the angiogenesis inhibitor endostatin that can inhibit endothelial migration in vitro and also inhibit endometriosis in a mouse model. Pharmacologic intervention in a surgically induced mouse model of endometriosis and endothelial migration assay. Animal research and laboratory facility. Eight-week-old, female C57BL/6 mice and human microvascular endothelial cells. Eight overlapping synthetic peptides were tested for inhibitory potential on endothelial migration in vitro. The peptides with significant activity then were given for 4 weeks to mice after implantation of autologous endometrium. Inhibition of vascular endothelial growth factor-induced endothelial migration for in vitro studies. In vivo studies examined the growth rate of endometriotic lesions after 4 weeks of treatment, as well as the effect on estrous cycling and ovulation as assessed by corpus luteum formation. The N-terminal mP-1 peptide and the internal mP-6 peptide inhibited endothelial migration in a dose-dependent manner. Additionally, both synthetic peptides suppressed growth of endometriotic lesions significantly in vivo. However, estrous cycling and corpus luteum formation were normal in both groups. Short endostatin fragments may be promising as a new, nontoxic therapeutic strategy for the treatment of endometriosis without inhibition of normal estrous cycles.

Membrane Type 1-Matrix Metalloproteinase Is Regulated by Chemokines Monocyte-Chemoattractant Protein-1/CCL2 and Interleukin-8/CXCL8 in Endothelial Cells during Angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.

Captopril suppresses Post-transplantation angiogenic activity in rat allograft coronary vessels

Background The development of transplant coronary artery disease is associated with neovascularization in the thickened neointima. We previously reported that captopril inhibits neointimal proliferation in a rat allograft model. We postulated that angiogenic inducers are upregulated post-transplantation and captopril ameliorates transplant coronary artery disease by suppressing the angiogenic activity of coronaries. Methods Animals received no treatment or captopril (50 mg/kg/day). Allograft hearts were analyzed at post-transplantation Days 0, 14, and 21 and angiogenic inducer, plasma platelet-activating factor, determined. The conditioned media from coronaries and myocardium were tested for vascular endothelial growth factor, thrombospondin-1 and angiogenic activity using an endothelial migration assay and rat corneal neovascularization assay. Results The captopril-treated group had reduced plasma platelet-activating factor and coronary media revealed earlier upregulation of thrombospondin-1 secretion, diminished vascular endothelial growth factor and no angiogenic activity. At Day 0, the coronary and myocardial conditioned medium had inhibitory activity due to thrombospondin-1, and circulating levels of platelet-activating factor were negligible. By 21 days post-transplantation, plasma platelet-activating factor was elevated and the conditioned medium from untreated coronaries had significantly higher angiogenic activity due to increased vascular endothelial growth factor whereas the myocardium remained non-angiogenic. Conclusions After transplantation, coronary vessels switch to an angiogenic phenotype and vascular endothelial growth factor contributes to the high angiogenic activity, possibly exacerbated by high circulating levels of platelet-activating factor. The ability of captopril to modulate angiogenic mediators and maintain the allograft coronary to its normal anti-angiogenic phenotype may be one mechanism by which it suppresses transplant coronary artery disease.