Low concentrations of TNF-α in vitro transform the phenotype of vascular smooth muscle cells and enhance their survival in a three-dimensional culture system.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are commonly used as seed cells in tissue-engineered vascular constructions. However, their variable phenotypes and difficult to control functions pose challenges. This study aimed to overcome these obstacles using a three-dimensional culture system. METHODS: Calf VSMCs were administered tumor necrosis factor-alpha (TNF-α) before culturing in two- and three-dimensional well plates and polyglycolic acid (PGA) scaffolds, respectively. The phenotypic markers of VSMCs were detected by immunofluorescence staining and western blotting, and the proliferation and migration abilities of VSMCs were detected by CCK-8, EDU, cell counting, scratch, and Transwell assays. RESULTS: TNF-α rapidly decreased the contractile phenotypic markers and elevated the synthetic phenotypic markers of VSMCs, as well as markedly increasing the proliferation and migration ability of VSMCs under two- and three-dimensional culture conditions. CONCLUSIONS: TNF-α can rapidly induce a phenotypic shift in VSMCs and change their viability on PGA scaffolds.

Insulin-transferrin-selenium promote formation of tissue-engineered vascular grafts in early stage of culture.

To create tissue-engineered vascular grafts (TEVGs) in vitro, vascular smooth muscle cells (VSMCs) must function effectively and produce sufficient extracellular matrix (ECM) in a three-dimensional space. In this study, we investigated whether the addition of insulin-transferrin-selenium (ITS), a medium supplement, could enhance TEVG formation. PGA fabric was used as the scaffold, and 1% ITS was added to the medium. After two weeks, the tissues were examined using electron microscopy and staining. The ITS group exhibited a denser structure and increased collagen production. VSMCs were cultured in two dimensions with ITS and assessed for collagen production, cell growth, and glucose metabolism. The results showed that ITS supplementation increased collagen production, cell growth, glucose utilization, lactate production, and ATP levels. Furthermore, reducing the amount of fetal bovine serum (FBS) in the medium did not affect the TEVGs or VSMCs when ITS was present. In conclusion, ITS improves TEVG construction by promoting VSMCs growth and reducing the need for FBS.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro.

BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype. PURPOSE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR). METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR. RESULTS: Notably, 225 µg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis. CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Macromolecular Crowding Enhances Matrix Protein Deposition in Tissue-Engineered Vascular Grafts.

Successful in vitro culture of small-diameter tissue-engineered vascular grafts (TEVGs) requires rapid deposition of biomacromolecules secreted by vascular smooth muscle cells in a polyglycolic acid mesh scaffold's three-dimensional (3D) porous environment. However, common media have lower crowding conditions than in vivo tissue fluids. In addition, during the early stages of construction, most of the biomolecules secreted by the cells into the medium are lost, which negatively affects the TEVG culture process. In this study, we propose the use of macromolecular crowding (MMC) to enhance medium crowding to improve the deposition and self-assembly efficiency of major biomolecules in the early stages of TEVG culture. The addition of carrageenan significantly increased the degree of MMC in the culture medium without affecting cell viability, proliferation, and metabolic activity. Protein analysis demonstrated that the deposition of collagen types I and III and fibronectin increased significantly in the cell layers of two-dimensional and 3D smooth muscle cell cultures after the addition of a MMC agent. Collagen type I in the culture medium decreased significantly compared with that in the medium without a MMC agent. Scanning electron microscopy demonstrated that MMC agents considerably enhanced the formation of matrix protein structures during the early stages of 3D culture. Hence, MMC modifies the crowding degree of the culture medium, resulting in the rapid formation of numerous matrix proteins and fiber structures. Impact Statement Small-diameter tissue-engineered vascular grafts (TEVGs) are one of the most promising means of treating cardiovascular diseases; however, the in vitro construction of TEVGs has some limitations, such as slow deposition of extracellular matrix (ECM), long culture period, and poor mechanical properties. We hypothesized that macromolecular crowding can increase the crowding of the culture medium to construct a more bionic microenvironment, which enhances ECM deposition in the medium to the cell layer and reduces collagen loss, accelerating and enhancing TEVG culture and construction in vitro.

PRP significantly promotes the adhesion and migration of vascular smooth muscle cells on stent material.

BACKGROUND: The adhesion and survival state of cells on scaffold material is a major problem in tissue-engineered blood vessel (TEBV) culture. Platelet-rich plasma (PRP) contains a large amount of biologically active factors and fibrin, which is expected to play an important role in TEBV culture. PURPOSE: To combine PRP with cells and scaffold material to promote cell adhesion and biological activity on the scaffold material. METHODS: The adhesion status and migration of SMCs under the optimal concentration suitable for SMC growth and the optimal concentration of PRP were examined by scanning electron microscopy, HE staining, CCK-8 assays, qPCR, WB, and other experimental methods and compared with those under the conventional culture (20% FBS); finally, the effect of PRP on the deposition of ECM in vascular tissue engineering culture was verified by three-dimensional culture. RESULTS: PRP at 20% is a suitable concentration for SMCs. Compared with the control group, the 20% PRP group had better migration, and the number of SMC adhesions was significantly higher than that of the control group. In addition, collagen deposition in the experimental group was significantly higher than that in the control group. CONCLUSION: PRP (20%) can promote SMC adhesion, migration, and collagen deposition on the scaffold material.

Long-term exposure to genistein inhibits the proliferation of gallbladder cancer by downregulating the MCM complex.

Soy isoflavones are natural tyrosine kinase inhibitors closely associated with decreased morbidity and mortality of various tumors. The activation of tyrosine kinases such as ERBB2 is the mechanism by which cholecystitis transforms into gallbladder cancer (GBC), therefore, it is important to investigate the relationship between long-term exposure to soy isoflavones and the occurrence and progression of GBC. This case-control study (n = 85 pairs) found that the high level of plasma soy isoflavone-genistein (GEN) was associated with a lower risk of gallbladder cancer (≥326.00 ng/mL compared to ≤19.30 ng/mL, crude odds ratio 0.15, 95% CI 0.04-0.59; P for trend = 0.016), and that the level of GEN exposure negatively correlated with Ki67 expression in GBC tissue (n = 85). Consistent with these results, the proliferation of GBC cells was inhibited in the long-term exposure models of GEN in vitro and in vivo. The long-term exposure to GEN reduced the tyrosine kinase activity of ERBB2 and impaired the function of the PTK6-AKT-GSK3ß axis, leading to downregulation of the MCM complex in GBC cells. In summary, long-term exposure to GEN associated with soy products intake might play a certain role in preventing GBC and even inhibiting the proliferation of GBC cells.