HtrA3 paves the way for MSC migration and promotes osteogenesis.

Mesenchymal stem cell (MSC) migration determines the healing capacity of bone and is crucial in promoting bone regeneration. Migration of MSCs is highly dependent on degradation of extracellular matrix by proteolytic enzymes. However, the underlying mechanisms of how enzymolysis paves the way for MSCs to migrate from their niche to the defect area is still not fully understood. Here, this study shows that high-temperature requirement A3 (HtrA3) overcomes the physical barrier and provides anchor points through collagen IV degradation, paving the way for MSC migration. HtrA3 is upregulated in MSCs at the leading edge of bone defect during the early stage of healing. HtrA3 degrades the surrounding collagen IV, which increases the collagen network porosity and increases integrin ß1 expression. Subsequently, integrin ß1 enhances the mechanotransduction of MSCs, thus remodeling the cytoskeleton, increasing cellular stiffness and nuclear translocation of YAP, eventually promoting the migration and subsequent osteogenic differentiation of MSCs. Local administration of recombinant HtrA3 in rat cranial bone defects significantly increases new bone formation and further validates the enhancement of MSC migration. This study helps to reveal the novel roles of HtrA3, explore potential targets for regenerative medicine, and offer new insights for the development of bioactive materials.

Matrix stiffness modulates tip cell formation through the p-PXN-Rac1-YAP signaling axis.

Endothelial tip cell outgrowth of blood-vessel sprouts marks the initiation of angiogenesis which is critical in physiological and pathophysiological procedures. However, how mechanical characteristics of extracellular matrix (ECM) modulates tip cell formation has been largely neglected. In this study, we found enhanced CD31 expression in the stiffening outer layer of hepatocellular carcinoma than in surrounding soft tissues. Stiffened matrix promoted sprouting from endothelial cell (EC) spheroids and upregulated expressions of tip cell-enriched genes in vitro. Moreover, tip cells showed increased cellular stiffness, more actin cytoskeleton organization and enhanced YAP nuclear transfer than stalk and phalanx ECs. We further uncovered that substrate stiffness regulates FAK and Paxillin phosphorylation in focal adhesion of ECs promoting Rac1 transition from inactive to active state. YAP is subsequently activated and translocated into nucleus, leading to increased tip cell specification. p-Paxillin can also loosen the intercellular connection which also facilitates tip cell specification. Collectively our present study shows that matrix stiffness modulates tip cell formation through p-PXN-Rac1-YAP signaling axis, shedding light on the role of mechanotransduction in tip cell formation. This is of special significance in biomaterial design and treatment of some pathological situations.

Cell Membrane Vesicles with Enriched CXCR4 Display Enhances Their Targeted Delivery as Drug Carriers to Inflammatory Sites.

Cell membrane vesicles (CMVs) are composed of natural cell membranes which makes them effective drug delivery systems with low immunogenicity and prolonged circulation time. However, targeting delivery of CMVs in vivo for clinical applications is still a major challenge. In this study, CXCR4 recombinant lentivirus is transfected into MC-3T3 cells and membrane CXCR4-enriched MC-3T3 cells are obtained. CMVs with enriched membrane CXCR4 display (CXCR4-CMVs) are obtained from the transfected MC-3T3 cells. Curcumin, an effective natural anti-inflammatory compound, is encapsulated into CXCR4-CMVs through physical entrapment (CXCR4/Cur-CMVs), with the membrane integrity of CXCR4/Cur-CMVs being well-preserved. CXCR4/Cur-CMVs induce enhanced M2 macrophage polarization, exhibit anti-inflammatory effects, and significantly improve homing via the CXCR4/CXCL12 axis in vitro. Utilizing ulcerative colitis and apical periodontitis as inflammatory disease models, it is found that CXCR4/Cur-CMVs are obviously aggregated within inflammatory areas after intravenous administration, which results in significant amelioration of ulcerative colitis and apical periodontitis. Therefore, this research may provide a feasible and innovative approach for fabricating an inflammatory site-targeting delivery system, by engineering CMVs to increase membrane-presenting CXCR4 receptor.

Three-dimensional radiographic and histological tracking of rat mandibular defect repair after inferior alveolar nerve axotomy.

OBJECTIVE: To sequentially track mandibular defect repair by using radiographic and histological techniques, so as to compare repair patterns of sensory denervated versus innervated mandibles. DESIGN: Forty Sprague-Dawley rats were subjected to unilateral inferior alveolar nerve (IAN) axotomy and bilateral 3 mm full-thickness circular osteotomy of their mandibles. Micro-CT and histological staining were applied to track the repair process of the mandibular defects at 1, 2, 4, and 8 weeks after surgery. RESULTS: The bone volume of both sides increased by 2 weeks post-operation, and then gradually decreased. The new bone volumes of the axotomy side were significantly less than that of the sham side at 1, 2, and 4 weeks post-surgery, whereas no significant differences were detected at 8 weeks post-surgery. Meanwhile, there were no significant differences in bone mineral density between the two sides during repair. Noteworthy, the repaired bone remained more vertically than horizontally aligned throughout the repair process. CONCLUSION: IAN axotomy decreases the quantity of bone calluses during the early stage of mandibular defect repair, but with no effect on the degree of mineralization. The shape of the defect area appeared to be aligned with the direction of local mechanical force produced by masticatory muscles.

The Selective RNA Polymerase I Inhibitor CX-5461 Mitigates Neointimal Remodeling in a Modified Model of Rat Aortic Transplantation.

BACKGROUND: Transplant vasculopathy is a major cause of chronic rejection of transplanted organs. In the present study, we examined the effects of CX-5461, a novel selective inhibitor of RNA polymerase I, on development of transplant vasculopathy using a modified model of rat aortic transplantation. METHODS: The thoracic aortas from Fischer rats were transplanted into the abdominal cavity of Lewis rats. CX-5461 was mixed in pluronic gel and administered via perivascular release. RESULTS: Treatment with CX-5461 mitigated the development of neointimal hyperplasia and vascular inflammation. This effect was likely to be attributable in part to inhibition of macrophage-dependent innate immunity reactions. Specifically, CX-5461 exhibited potent inhibitory effects on macrophage migration and lipopolysaccharide-induced activation. Treatment with CX-5461 also prevented macrophage differentiation and maturation from primary bone marrow cells. In macrophages, CX-5461 did not alter the total amount of p53 protein, but significantly increased p53 phosphorylation, which was involved in regulating cytokine-stimulated macrophage proliferation. CONCLUSIONS: In conclusion, our results suggest that pharmacological inhibition of RNA polymerase I may be a novel strategy to treat transplantation-induced arterial remodeling.

Modulating Surface Potential by Controlling the ß Phase Content in Poly(vinylidene fluoridetrifluoroethylene) Membranes Enhances Bone Regeneration.

Bioelectricity plays a vital role in living organisms. Although electrical stimulation is introduced in the field of bone regeneration, the concept of a dose-response relationship between surface potential and osteogenesis is not thoroughly studied. To optimize the osteogenic properties of different surface potentials, a flexible piezoelectric membrane, poly(vinylidene fluoridetrifluoroethylene) [P(VDF-TrFE)], is fabricated by annealing treatment to control its ß phases. The surface potential and piezoelectric coefficients (d33 ) of the membranes can be regulated by increasing ß phase contents. Compared with d33  = 20 pC N-1 (surface potential = -78 mV) and unpolarized membranes, bone marrow mesenchymal stem cells (BM-MSCs) cultured on the d33  = 10 pC N-1 (surface potential = -53 mV) membranes have better osteogenic properties. In vivo, d33  = 10 pC N-1 membranes result in rapid bone regeneration and complete mature bone-structure formation. BM-MSCs on d33  = 10 pC N-1 membranes have the lowest reactive oxygen species level and the highest mitochondrial membrane electric potential, implying that these membranes provide the best electrical qunantity for BM-MSCs' proliferation and energy metabolism. This study establishes an effective method to control the surface potential of P(VDF-Trfe) membranes and highlights the importance of optimized electrical stimulation in bone regeneration.

Early adventitial activation characterized by NADPH oxidase expression and neovascularization in an aortic transplantation model.

Increasing evidence has suggested that arterial adventitia may contribute to pathological vessel remodeling by producing reactive oxygen species and promoting neovascularization. However, these processes have not been studied yet in transplantation-induced vascular pathologies. We characterized the dynamic changes in NADPH oxidase expression and adventitial angiogenic response in a model of allograft aortic transplantation. The thoracic aorta from Fischer 344 rats were transplanted into the abdominal aorta of Lewis rats. Graft specimens were collected on days 0.5, 3, 7, and 14 for morphometry, immunohistochemistry, immunofluorescence staining, and quantitative PCR tests. Following transplantation, adventitial thickening was found as early as day 3, while neointima was observed from day 7. As compared to normal adventitial tissue, the expression levels of NADPH oxidase subunits gp91phox and p47phox in graft adventitia were elevated from day 3 and further increased up to day 14. Immunohistochemistry staining showed that infiltrating macrophages appeared to be a major source of NADPH oxidase expression. Increases in NADPH oxidase expression were also detected in fibroblasts isolated from the graft adventitia. Gene silencing of p47phox significantly suppressed proliferation and migration of the graft fibroblast cells. We also showed that adventitial thickening was accompanied by increased adventitial neovascularization; at day 14, there was a positive correlation between the density of adventitial microvessels and the neointimal thickness. Transplantation injury induces NADPH oxidase expression and neovascularization in the adventitia, raising the possibility that the activated adventitia may represent a target site for prevention of transplantation-induced transplant vasculopathy.

KIF2A silencing inhibits the proliferation and migration of breast cancer cells and correlates with unfavorable prognosis in breast cancer.

BACKGROUND: Kinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied. METHODS: Immunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients' survival in relation to KIF2A expression was estimated using the Kaplan-Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection. RESULTS: The expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P < 0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P < 0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P < 0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P < 0.05). CONCLUSIONS: KIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer.

Correlation of inflammatory cells in adventitia and formation and extending of atherosclerotic lesions in coronary artery of apolipoprotein E gene knockout mice.

Accumulating evidence shows that adventitial inflammation contributes to the development of atherosclerotic lesions. The aim of this study was to investigate the relationship between atherosclerotic lesions in coronary artery (CA) and accumulation of inflammatory cells at local adventitia in apolipoprotein E gene knockout (apoE-/-) mice. Modified Movat's pentachrome staining, HE staining, immunohistochemistry and transmission electron microscopy were used to observe and to identify serial paraffin sections of aortic foot and inflammatory cells in CA adventitia of apoE-/- mice of 60 weeks old. There was always accumulation of inflammatory cells in the adventitia of CA with extending lesions from aortic orifice to CA trunks. The CA samples were divided into type I: infiltration of inflammatory cells in CA adventitia without lesions extending in the intima, type II: infiltration of inflammatory cells in CA adventitia with the top of extending lesions in the intima and type III: infiltration of inflammatory cells at CA adventitia with lesions covering all the face of intima. The three types of CA sample represent the different developmental processes of atherosclerotic lesions, respectively. No extending lesions were found in the CA trunks without inflammatory cells in adventitia. In type I samples, 60% of infiltrated inflammatory cells were macrophages 57% of infiltrated cells were neutrophils in type II samples; 67% of infiltrated cells were lymphocytes in type III samples. Our studies revealed that adventitial inflammation may be an early event in the development of atherosclerotic lesions. Different cell types predominate in different stages of CA adventitia. The neutrophils are closely related to the extending of atherosclerotic lesions.