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.

A novel role of angiotensin II in epidermal cell lineage determination: Angiotensin II promotes the differentiation of mesenchymal stem cells into keratinocytes through the p38 MAPK, JNK and JAK2 signalling pathways.

BACKGROUND: Recent evidence suggests that angiotensin II (Ang II) plays a role in cutaneous wound healing. Mesenchymal stem cells (MSCs) are known as a rich source of cells that re-establish healed skin. However, the potential impact of Ang II on MSC differentiation into keratinocytes is still unknown. OBJECTIVE: The present study was conducted to explore the effect of Ang II on the differentiation of bone marrow-derived MSCs (BM-MSCs) into keratinocytes. METHODS: Bone marrow-derived MSCs were isolated from rat bone marrow and cultured. The expression of Ang II type 1 (AT1 ) and type 2 (AT2 ) receptors was examined by immunofluorescence staining. The differentiation of BM-MSCs into keratinocytes was investigated by flow cytometry or/and histological observation. RESULTS: The BM-MSCs constitutively expressed both AT1 and AT2 receptors. The differentiation of BM-MSCs into keratinocytes was successfully induced. Interestingly, incubation of BM-MSCs with Ang II further promoted the differentiation of BM-MSCs into keratinocyte, which was abolished by pretreament with losartan, an AT1 receptor antagonist, but not by PD123319, an AT2 receptor antagonist. Moreover, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the Janus-activated kinase (JAK)2 inhibitor AG490 suppressed Ang II-induced differentiation of BM-MSCs into keratinocytes. The phosphoinositide-3 kinase (PI3K) inhibitor wortmannin and MEK1/2 inhibitor U0126 had no effect on BM-MSC differentiation into keratinocytes. CONCLUSIONS: Our data demonstrated for the first time that Ang II plays a promotive role in the differentiation of BM-MSC into keratinocytes through the AT1 receptor, and that the p38 MAPK, JNK and JAK2 signalling pathways are involved in this process.

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.

Retraction of "lncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells".

[This retracts the article DOI: 10.1515/med-2021-0406.].

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.

lncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells.

Gastric cancer (GC) is one of the most common malignancies in digestive system. Accumulating evidence reveals the critical role of long noncoding RNAs (lncRNAs) in GC development. The study aimed to explore the functions and mechanism of lncRNA actin alpha 2, smooth muscle antisense RNA 1 (ACTA2-AS1) in GC. Reverse transcription-quantitative polymerase chain reaction analyses and subcellular fractionation assays showed that ACTA2-AS1 was lowly expressed in GC cells and was mainly distributed in the cytoplasm. Overexpressed ACTA2-AS1 inhibited GC cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition process, as suggested by cell counting kit-8 assays, colony formation assays, wound healing assays, Transwell assays and Western blot analyses. Mechanistically, ACTA2-AS1 served as a competing endogenous RNA (ceRNA) to bind with miR-378a-3p and thereby, antagonized the inhibitory effect of miR-378a-3p on the expression of messenger RNA phosphatidylinositol specific phospholipase C X domain containing 2 (PLCXD2). The binding capacity between miR-378a-3p and ACTA2-AS1 (or PLCXD2) was detected by RNA pulldown assays, luciferase reporter assays and RNA immunoprecipitation assays. Moreover, PLCXD2 knockdown rescued the inhibitory effect of ACTA2-AS1 overexpression on malignant behaviors of GC cells. Overall, ACTA2-AS1 inhibits malignant phenotypes of GC cells by acting as a ceRNA to target miR-378a-3p/PLCXD2 axis.

Autologous fat transplantation for the treatment of abdominal wall scar adhesions after cesarean section.

Cesarean section results in scarring, which usually leads to adhesion between the subcutaneous fat and the abdominal wall muscle. The present study aimed to evaluate the therapeutic effect of autologous fat grafting on scar adhesion to the abdominal wall after cesarean section. Thirty-six patients with scar adhesion to the abdominal wall after cesarean section were recruited and treated between October 2013 and December 2015. The adhesion between the subcutaneous fat and the abdominal wall muscle was carefully separated through a small incision in the original scar to form multiple subcutaneous tunnels. Aspirated fat was injected into the scar lesion and subcutaneous tunnels, and the wound was then sutured. The clinical outcome was evaluated by comparing the pretreatment and 1-year posttreatment photographs and Patient and Observer Scar Assessment Scale (POSAS) scores. All patients had a marked improvement in the appearance, texture, and depression of the scar during 12 months of follow-up. The 1-year posttreatment POSAS scores for the color, pain, pruritus, hardness, fullness, mobility, and appearance of the scar were significantly decreased compared with the pretreatment scores. Hematoxylin-eosin staining revealed adipocyte-like cells in treated scar tissue specimens obtained 1 year after treatment. None of the patients reported severe adverse reactions. Autologous fat grafting combined with adhesion release may be a good treatment option for abdominal wall scarring after cesarean section. This method is minimally invasive and effective in achieving good functional and esthetic outcomes.

Antiaging Properties of Exosomes from Adipose-Derived Mesenchymal Stem Cells in Photoaged Rat Skin.

Adipose-derived stem cells (ADSCs) have been documented as possible candidates for skin rejuvenation. However, the effects of ADSC-derived exosomes on photoaged skin remain to be fully elucidated. This study was aimed at determining the antiaging effects of ADSC-derived exosomes on photoaged skin. Human ADSCs were isolated from the adipose tissue of healthy women and cultured in vitro. Then, exosomes were extracted from the cultured ADSCs, purified by ultracentrifugation, and verified by examination of cell morphology using transmission electron microscopy and the identification of specific biomarkers. Meanwhile, the optimal exosome concentration and treatment time were selected. The photoaged skin model was created by subjecting Sprague-Dawley rats to ultraviolet B radiation. Exosomes were injected into the photoaged skin in a single therapeutic dose. The thickness of the epidermis and dermis was observed by HE staining. The relative mRNA expression of type I collagen, type III collagen, and matrix metalloproteinases (MMP-1 and MMP-3) was determined by real-time PCR. In the rat model of photoaged skin, the injected exosomes markedly decreased the epidermal thickness and increased the dermal thickness of the photoaged skin 7 days after treatment. Moreover, the proportion of the stratum corneum of the epidermis was decreased. Furthermore, real-time RT-PCR showed that the mRNA expression of type I collagen was increased and that of type III collagen, MMP-1, and MMP-3 was decreased. Our results demonstrate that ADSC-derived exosome treatment could significantly improve skin photodamage and that ADSC-derived exosomes may be a potential agent for photoaged skin treatment.

Effects of Carbon Arc Lamp Irradiation on Wound Healing in a Rat Cutaneous Full-Thickness Wound Model.

Objective: The objective of the present study was to investigate the application of a carbon arc lamp on wound healing in a rat cutaneous full-thickness wound model. Background data: In clinical practice, wound healing has been promoted by irradiation with a carbon arc lamp. However, the corresponding mechanism has not been clearly defined. Methods: A cutaneous full-thickness wound on the back of rats was irradiated using a carbon arc lamp at a wavelength peak range of 620-740 nm with 54 J/cm2. Injured sham-irradiated control rats were used as the control. The rats were euthanized after 7, 14, and 21 days, while wound reepithelialization and healing quality were examined by histological analyses with comparison between groups. Cell proliferation was observed by 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining. Results: Irradiation by the carbon arc lamp significantly accelerated wound healing. The wound-healing rate in the treated group at day 21 was 98.42% ± 0.56%, compared with 93.58% ± 1.26% in the control group (p < 0.05). Significant increases in the length of epithelial edges, collagen content, and microvessel density were observed in the wound sites in the treated group at days 7, 14, and 21 (p < 0.05). Moreover, the number of BrdU-labeled cells increased in the wound edge at days 7 and 14 due to irradiation (p < 0.05). Conclusions: The results demonstrated that the carbon arc lamp can promote wound healing together with improvement in its quality by stimulating cell proliferation.

Effects of storage culture media, temperature and duration on human adipose­derived stem cell viability for clinical use.

Adipose­derived stem cells (ADSCs) are mesenchymal stem cells that are often used in regenerative medicine. Maintaining ADSC viability is important, as this optimizes the curative effects of cell therapy. However, the optimal conditions for cell viability preservation remain unknown. The present study aimed to acquire a better protocol for ADSC storage by comparing the effects of various solutions and temperatures for ADSC preservation, in order to suggest the most effective methods of short­term ADSC preservation for clinical use. ADSCs from passage 2 were suspended in solutions comprising 0.9% NaCl, 10% human serum (HS) or 10% platelet­rich plasma (PRP). Suspended cells were maintained at 4˚C or room temperature (~26˚C) for 2, 4 and 6 h. The differentiation capacity, apoptosis and proliferation of ADSCs were determined by oil red O/alizarin red S staining, flow cytometry, and a cell counting kit­8 cell proliferation assay, respectively. In addition, reverse transcription­quantitative polymerase chain reaction and western blot analysis was performed. The results revealed that proliferation of ADSCs decreased with time. The optimal time for ADSC use was ~2 h, and 4 h was determined to be the latest time that ADSCs should be used. The 10% HS group had the highest survival rate, followed by the 10% PRP group; these two groups had higher survival rates than the 0.9% NaCl group (P<0.05). HS and PRP at 4˚C enhanced the ADSC proliferation rate (P<0.05), although the difference between these two groups was insignificant (P>0.05). In conclusion, the optimal time to use ADSCs was <2 h, and should not exceed 4 h. It was recommended that, for the transportation and short­term storage of ADSCs during clinical use, they should be stored with 10% HS at 4˚C to maintain ADSC viability. In addition, this was a cost­effective and safe method.

Effects of Carbon Arc Lamp Irradiation on Wound Healing in a Rat Cutaneous Full-Thickness Wound Model.

OBJECTIVE: The objective of the present study was to investigate the application of a carbon arc lamp on wound healing in a rat cutaneous full-thickness wound model. BACKGROUND DATA: In clinical practice, wound healing has been promoted by irradiation with a carbon arc lamp. However, the corresponding mechanism has not been clearly defined. METHODS: A cutaneous full-thickness wound on the back of rats was irradiated using a carbon arc lamp at a wavelength peak range of 620-740 nm with 54 J/cm2. Injured sham-irradiated control rats were used as the control. The rats were euthanized after 7, 14, and 21 days, while wound reepithelialization and healing quality were examined by histological analyses with comparison between groups. Cell proliferation was observed by 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining. RESULTS: Irradiation by the carbon arc lamp significantly accelerated wound healing. The wound-healing rate in the treated group at day 21 was 98.42% ± 0.56%, compared with 93.58% ± 1.26% in the control group (p < 0.05). Significant increases in the length of epithelial edges, collagen content, and microvessel density were observed in the wound sites in the treated group at days 7, 14, and 21 (p < 0.05). Moreover, the number of BrdU-labeled cells increased in the wound edge at days 7 and 14 due to irradiation (p < 0.05). CONCLUSIONS: The results demonstrated that the carbon arc lamp can promote wound healing together with improvement in its quality by stimulating cell proliferation.