• Biomolecules & Therapeutics
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• 제26권5호
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• pp.474-480
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• 2018

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 in vitro 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 $IC_{50}$ of vatalanib in ECFC+MSC spheroids at 24 h was $4.0{\pm}0.40{\mu}M$, which are more correlated with the data of previous animal studies when compared with ECFC spheroids ($0.2{\pm}0.03{\mu}M$). 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 in vitro assay model to evaluate pro- or anti-angiogenic property.

• The Korean Journal of Physiology and Pharmacology
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• 제18권2호
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• pp.163-168
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• 2014

Endothelial progenitor cells (EPCs) are known to play an important role in the repair of damaged blood vessels. We used an endothelial progenitor cell colony-forming assay (EPC-CFA) to determine whether EPC numbers could be increased in healthy individuals through regular exercise training. The number of functional EPCs obtained from human peripheral blood-derived AC133 stem cells was measured after a 28-day regular exercise training program. The number of total endothelial progenitor cell colony-forming units (EPC-CFU) was significantly increased compared to that in the control group (p=0.02, n=5). In addition, we observed a significant decrease in homocysteine levels followed by an increase in the number of EPC-CFUs (p=0.04, n=5), indicating that the 28-day regular exercise training could increase the number of EPC colonies and decrease homocysteine levels. Moreover, an inverse correlation was observed between small-endothelial progenitor cell colony-forming units (small-EPC-CFUs) and plasma homocysteine levels in healthy men (r=-0.8125, p=0.047). We found that regular exercise training could increase the number of EPC-CFUs and decrease homocysteine levels, thus decreasing the cardiovascular disease risk in men.

• The Korean Journal of Physiology and Pharmacology
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• 제22권6호
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• pp.705-712
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• 2018

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). In vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro 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 $2{\times}2$ stitched area from the $4{\times}$ 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, $IC_{50}$ 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.

• Journal of Korean Neurosurgical Society
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• 제66권6호
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• pp.642-651
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• 2023

Objective : Endothelial colony-forming cells (ECFCs) have been reported to play an important role in the pathogenesis of moyamoya disease (MMD). We have previously observed stagnant growth in MMD ECFCs with functional impairment of tubule formation. We aimed to verify the key regulators and related signaling pathways involved in the functional defects of MMD ECFCs. Methods : ECFCs were cultured from peripheral blood mononuclear cells of healthy volunteers (normal) and MMD patients. Low-density lipoproteins uptake, flow cytometry, high content screening, senescence-associated β-galactosidase, immunofluorescence, cell cycle, tubule formation, microarray, real-time quantitative polymerase chain reaction, small interfering RNA transfection, and western blot analyses were performed. Results : The acquisition of cells that can be cultured for a long time with the characteristics of late ECFCs was significantly lower in the MMD patients than the normal. Importantly, the MMD ECFCs showed decreased cellular proliferation with G1 cell cycle arrest and cellular senescence compared to the normal ECFCs. A pathway enrichment analysis demonstrated that the cell cycle pathway was the major enriched pathway, which is consistent with the results of the functional analysis of ECFCs. Among the genes associated with the cell cycle, cyclin-dependent kinase inhibitor 2A (CDKN2A) showed the highest expression in MMD ECFCs. Knockdown of CDKN2A in MMD ECFCs enhanced proliferation by reducing G1 cell cycle arrest and inhibiting senescence through the regulation of CDK4 and phospho retinoblastoma protein. Conclusion : Our study suggests that CDKN2A plays an important role in the growth retardation of MMD ECFCs by inducing cell cycle arrest and senescence.

• Biomolecules & Therapeutics
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• 제20권2호
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• pp.158-164
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• 2012

Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using $CD34^+$ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using $CD34^+$ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including $CD34^+$, $CD34^+/CD133^+$, $CD34^+/CD31^+$ and $CD34^+/CXCR4^+$. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis.

• Journal of Genetic Medicine
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• 제7권2호
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• pp.138-144
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• 2010

목 적: 자궁내막의 신혈관 형성을 조정하는 혈관내피전구세포는 자궁-태반 순환에서의 신혈관 형성에 관여하는 것으로 알려져 있다. 혈관내피전구세포의 정량적 지표로 사용되는 집락생성단위(CFU)의 감소는 혈관질환의 예측 표지자로서 보고되고 있다. 본 연구에서는 정상 산모와 자간전증 산모 간에 CFU의 수적인 차이를 비교해 보고자 하였다. 대상 및 방법: 임신말기의 단태 정상 산모 26명과 자간전증산모 20명을 대상으로 혈관내피전구세포의 수를 CFU로 정량화하였다. 효소면역분석법을 이용하여 혈장내의 VEGF와 sFlt-1, PlGF 농도를 분석하였다. 결 과: CFU의 수는 정상 산모에 비해 자간전증 산모에서 매우 감소하였다[median value: 3 (range: 1-12) vs median value: 31(range:3-81) CFU/ well, P<0.001). 집락을 구성하는 대부분의 세포는 혈관내피세포의 특징적인 성질을 보였다(Ulex europaeus lectin의 발현 및 acetylated lowdensity lipoprotein의 섭취). 혈장내의 sFlt-1 농도는 정상 산모에 비해 자간전증 산모에서 매우 높은 반면(P<0.001), PlGF의 농도는 매우 낮았으며(P=0.004), sFlt-1과 PlGF의 농도는 CFU의 수와 연관성이 없었다. 결 론: 산모 말초혈액내의 CFU의 수적 감소가 자간전증 발생에 기여할 것으로 생각된다.

• International Journal of Oncology
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• 제54권4호
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• pp.1327-1336
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• 2019

Endothelial progenitor cells (EPCs) are bone marrow (BM)-derived progenitor cells that can differentiate into mature endothelial cells, contributing to vasculogenesis in the blood vessel formation process. Runt-related transcription factor 3 (RUNX3) belongs to the Runt domain family and is required for the differentiation of specific immune cells and neurons. The tumor suppressive role of RUNX3, via the induction of apoptosis and cell cycle arrest in a variety of cancers, and its deletion or frequent silencing by epigenetic mechanisms have been studied extensively; however, its role in the differentiation of EPCs is yet to be investigated. Therefore, in the present study, adult BM-derived hematopoietic stem cells (HSCs) were isolated from Runx3 heterozygous (Rx3^+/-) or wild-type (WT) mice. The differentiation of EPCs from the BM-derived HSCs of Rx3^+/- mice was found to be significantly increased compared with those of the WT mice, as determined by the number of small or large colony-forming units. The migration and tube formation abilities of Rx3^+/- EPCs were also observed to be significantly increased compared with those of WT EPCs. Furthermore, the number of circulating EPCs, defined as CD34⁺/vascular endothelial growth factor receptor 2 (VEGFR2)⁺ cells, was also significantly increased in Rx3^+/- mice. Hypoxia-inducible factor (HIF)-1α was upregulated in Rx3^+/- EPCs compared with WT EPCs, even under normoxic conditions. Furthermore, in a hindlimb ischemic mouse models, the recovery of blood flow was observed to be highly stimulated in Rx3^+/- mice compared with WT mice. Also, in a Lewis lung carcinoma cell allograft model, the tumor size in Rx3^+/- mice was significantly larger than that in WT mice, and the EPC cell population (CD34⁺/VEGFR2⁺ cells) recruited to the tumor was greater in the Rx3^+/- mice compared with the WT mice. In conclusion, the present study revealed that Runx3 inhibits vasculogenesis via the inhibition of EPC differentiation and functions via the suppression of HIF-1α activity.

• Journal of Gastric Cancer
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• 제18권4호
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• pp.356-367
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• 2018

Purpose: Kallikrein (KLK) proteases are hormone-like signaling molecules with critical functions in different cancers. This study investigated the expression of KLK6 in gastric cancer and its potential role in the growth, migration, and invasion of gastric cancer cells. Materials and Methods: In this study, we compared protein levels of KLK6, vascular endothelial growth factor (VEGF), and matrix metallopeptidase (MMP) 9 in normal gastric epithelial and gastric cancer cell lines by western blot. Fluorescence-activated cell sorting was employed to sort 2 clones of SGC-7901 cells with distinct KLK6 expression, namely, KLK6-high ($KLK6^{high}$) and KLK6-low ($KLK6^{low}$), which were then expanded. Lastly, immunohistochemical analysis was performed to investigate KLK6 expression in gastric cancer patients. Results: The expression levels of KLK6, VEGF, and MMP 9, were significantly higher in the gastric cancer cell lines SGC-7901, BGC-823, MKN-28, and MGC-803 than in the normal gastric epithelial cell line GES-1. Compared to $KLK6^{low}$ cells, $KLK6^{high}$ cells showed enhanced viability, colony-forming ability, migration, and invasion potential in vitro. Importantly, immunohistochemical analysis of a human gastric cancer tissue cohort revealed that the staining for KLK6, VEGF, and MMP9 was markedly stronger in the cancerous tissues than in the adjacent normal tissues. KLK6 expression also correlated with that of VEGF and MMP9 expression, as well as several key clinicopathological parameters. Conclusions: Together, these results suggest an important role for KLK6 in human gastric cancer progression.

• 한국축산식품학회지
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• 제44권1호
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• pp.204-215
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• 2024

This study was designed to examine the effect of Lactilactobacillus curvatus LB-P9 on hair regeneration. The treatment of LB-P9 conditioned medium increased the proliferation of both hair follicle dermal papilla cells and hair germinal matrix cells (hGMCs). Moreover, the expression levels of hair growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 7 were significantly elevated in hGMCs co-cultured with LB-P9. After time-synchronized depilation, mice were orally administered with either 4×10⁷ colony forming unit (CFU) of LB-P9 (low dose) or 4×10⁸ CFU of LB-P9 (high dose), once daily for 4 weeks. Compared with the vehicle (phosphate-buffered saline)-administrated group, the LB-P9-treated groups exhibited accelerated hair regrowth rate and enhanced hair thickness in a dose-dependent manner. Supporting this observation, both hair follicle numbers and the dermal thickness in skin tissues of the LB-P9-treated groups were increased, compared to those of the vehicle-treated group. These results might be explained by the increased level of β-catenin and number of hair follicle stem cells (CD34⁺ CD49f⁺ cells) in the skin tissues of mice administered with LB-P9, compared to the vehicle-treated mice. Also, increased serum levels of hair growth factors such as VEGF and insulin-like growth factor-1, and superoxide dismutase were found in the LB-P9-treated groups, compared to those of the vehicle-treated group. Taken together, these results might demonstrate that the oral administration of LB-P9 promotes hair regeneration by the enhancement of dermal papilla proliferation through the stimulation of hair growth factor production.

• BMB Reports
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• 제55권4호
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• pp.175-180
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• 2022

Peptides are gaining substantial attention as therapeutics for human diseases. However, they have limitations such as low bioavailability and poor pharmacokinetics. Periostin, a matricellular protein, can stimulate the repair of ischemic tissues by promoting angiogenesis. We have previously reported that a novel angiogenic peptide (amino acids 142-151) is responsible for the pro-angiogenic activity of periostin. To improve the in vivo delivery efficiency of periostin peptide (PP), we used proteins self-assembled into a hollow cage-like structure as a drug delivery nanoplatform in the present study. The periostin peptide was genetically inserted into lumazine synthase (isolated from Aquifex aeolicus) consisting of 60 identical subunits with an icosahedral capsid architecture. The periostin peptide-bearing lumazine synthase protein cage nanoparticle with 60 periostin peptides multivalently displayed was expressed in Escherichia coli and purified to homogeneity. Next, we examined angiogenic activities of this periostin peptide-bearing lumazine synthase protein cage nanoparticle. AaLS-periostin peptide (AaLS-PP), but not AaLS, promoted migration, proliferation, and tube formation of human endothelial colony-forming cells in vitro. Intramuscular injection of PP and AaLS-PP increased blood perfusion and attenuated severe limb loss in the ischemic hindlimb. However, AaLS did not increase blood perfusion or alleviate tissue necrosis. Moreover, in vivo administration of AaLS-PP, but not AaLS, stimulated angiogenesis in the ischemic hindlimb. These results suggest that AaLS is a highly useful nanoplatform for delivering pro-angiogenic peptides such as PP.