Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Duksung Women's University

Cell-based therapies to restore heart function after infarction have been tested in pre-clinical models and clinical trials with mixed results, and will likely require both contractile cells and a vascular network to support them. We and others have shown that human endothelial colony forming cells (ECFC) combined with mesenchymal progenitor cells (MPC) can be used to "bio-engineer" functional human blood vessels. Here we investigated whether ECFC + MPC form functional vessels in ischemic myocardium and whether this affects cardiac function or remodeling. Myocardial ischemia/reperfusion injury (IRI) was induced in 12-week-old immunodeficient rats by ligation of the left anterior descending coronary artery. After 40 min, myocardium was reperfused and ECFC + MPC (2 × 10(6) cells, 2:3 ratio) or PBS was injected. Luciferase assays after injection of luciferase-labeled ECFC + MPC showed that 1,500 ECFC were present at day 14. Human ECFC-lined perfused vessels were directly visualized by femoral vein injection of a fluorescently-tagged human-specific lectin in hearts injected with ECFC + MPC but not PBS alone. While infarct size at day 1 was no different, LV dimensions and heart weight to tibia length ratios were lower in cell-treated hearts compared with PBS at 4 months, suggesting post-infarction remodeling was ameliorated by local cell injection. Fractional shortening, LV wall motion score, and fibrotic area were not different between groups at 4 months. However, pressure-volume loops demonstrated improved cardiac function and reduced volumes in cell-treated animals. These data suggest that myocardial delivery of ECFC + MPC at reperfusion may provide a therapeutic strategy to mitigate LV remodeling and cardiac dysfunction after IRI.

Human vasculogenic cells form functional blood vessels and mitigate adverse remodeling after ischemia reperfusion injury in rats.

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death. 

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Endothelium-derived relaxing factors (EDRFs), including nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), play pivotal roles in regulating vascular tone. Reduced EDRFs cause impaired endothelium-dependent vasorelaxation, or endothelial dysfunction. Impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh) is consistently observed in conduit vessels in human patients and experimental animal models of hypertension. Because small resistance arteries are known to produce more than one type of EDRF, the mechanism(s) mediating endothelium-dependent vasorelaxation in small resistance arteries may be different from that observed in conduit vessels under hypertensive conditions, where vasorelaxation is mainly dependent on NO. EDHF has been described as one of the principal mediators of endothelium-dependent vasorelaxation in small resistance arteries in normotensive animals. Furthermore, EDHF appears to become the predominant endothelium-dependent vasorelaxation pathway when the endothelial NO synthase (NOS3)/NO pathway is absent, as in NOS3-knockout mice, whereas some studies have shown that the EDHF pathway is dysfunctional in experimental models of hypertension. This article reviews our current knowledge regarding EDRFs in small arteries under normotensive and hypertensive conditions. 

Endothelium-derived Relaxing Factors of Small Resistance Arteries in Hypertension.

Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.

Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle.

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an  3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the IC of vatalanib in ECFC+MSC spheroids at 24 h was 4.0 ± 0.40 µM, which are more correlated with the data of previous animal studies when compared with ECFC spheroids (0.2 ± 0.03 µM). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical  assay model to evaluate pro- or anti-angiogenic property.

Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells.

Many studies reported reduced antioxidant capacity in the vasculature under hypertensive conditions. However, little is known about the effects of antihypertensive treatments on the regulation of vascular antioxidant enzymes. Thus, we hypothesized that antihypertensive treatments prevent the reduction of antioxidant enzyme activity and expression in the small vessels of angiotensin II-induced hypertensive rats (ANG). We observed the small mesenteric arteries and small renal vessels of normotensive rats (NORM), ANG, and ANG treated with a triple antihypertensive therapy of reserpine, hydrochlorothiazide, and hydralazine (ANG + TTx). Systolic blood pressure was increased in ANG, which was attenuated by 2 weeks of triple therapy (127, 191, and 143 mmHg for NORM, ANG, and ANG + TTx, respectively;  < 0.05). Total superoxide dismutase (SOD) activity in the small mesenteric arteries of ANG was lower than that of NORM. The protein expression of SOD1 was lower in ANG than in NORM, whereas SOD2 and SOD3 expression was not different between the groups. Reduced SOD activity and SOD1 expression in ANG was not restored in ANG + TTx. Both SOD activity and SOD isoform expression in the small renal vessels of ANG were not different from those of NORM. Interestingly, SOD activity in the small renal vessels was reduced by TTx. Between groups, there was no difference in catalase activity or expression in both the small mesenteric arteries and small renal vessels. In conclusion, SOD activity in the small mesenteric arteries decreased by angiotensin II administration, but not by hypertension, which is caused by decreased SOD1 expression.

Superoxide Dismutase Activity in Small Mesenteric Arteries Is Downregulated by Angiotensin II but Not by Hypertension.

The tube formation assay is a widely used  experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs).  experimental results are crucial when considered the advisability of moving forward to  studies. Thus, the additional attentions to the  assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

Kyu-Tae Kang

College of Pharmacy,
Duksung Innovative Drug Center,
College of Pharmacy, Duksung Innovative Drug Center

Duksung Women's University

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Kyu-Tae Kang

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Duksung Women's University

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

College of Pharmacy,
Duksung Innovative Drug Center,
College of Pharmacy, Duksung Innovative Drug Center