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.

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.

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.

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.

[Design and verification of Luo-Ye pump-based stress formation for cultivation of tissue-engineered blood vessel].

OBJECTIVE: To improve Luo-Ye pump-based stress-forming system and optimize the stimulating effect on smooth muscle cells during cultivation of tissue-engineered blood vessels (TEBV). METHODS: A new Luo-Ye pump-based TEBV 3D culture system was developed by adding an air pump to the output of the bioreactor. A pressure guide wire was used to measure the stress at different points of the silicone tube inside the TEBV bio-reactor, and fitting curves of the stress changes over time was created using Origin 8.0 software. The TEBVs were constructed by seeding vascular smooth muscle cells (VSMCs) isolated from human umbilical artery on polyglycolic acid (PGA) and cultured under dynamic conditions with 40 mmHg resistance (improved group), dynamic conditions without resistance (control group) or static condition (static group) for 4 weeks. The harvested TEBVs were then examined with HE staining, masson staining, α-SMA immunohistochemical staining, and scanning and transmission electron microscopy with semi-quantitative analysis of collagen content and α-SMA expression. RESULTS: The measured stress values and the fitting curves showed that the stress stimuli from the Luo-Ye pump were enhanced by adding an air pump to the output of the bioreactor. Histological analysis revealed improved VSMC density, collagen content and α-SMA expression in the TEBVs constructed with the improved method as compared with those in the control and static groups. CONCLUSION: Adding an air pump to the Luo-Ye pump significantly enhances the stress stimulation in the TEBV 3-D culture system to promote the secretion function of VSMCs.

Synthetic peptide studies on the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein: perspective for SARS vaccine development.

BACKGROUND: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein. METHODS: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy. RESULTS: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788-820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This suggested that our S5 and S6 peptides may represent two minimum biologically active sequences of the immunogenic regions of the SARS-CoV S protein. CONCLUSIONS: Synthetic peptides can elicit specific antibodies to SARS-CoV. The study provides insights for the future development of SARS vaccine via the synthetic-peptide-based approach.