Inhibition of CSPG-PTPσ Activates Autophagy Flux and Lysosome Fusion, Aids Axon and Synaptic Reorganization in Spinal Cord Injury.

Chondroitin sulfate proteoglycans (CSPGs) and proteoglycan receptor protein tyrosine phosphatase σ (PTPσ) play a critical role in the pathology of spinal cord injury (SCI). CSPGs can be induced by autophagy inhibition in astrocyte. However, CSPG's impact on autophagy and its role in SCI is still unknown. We investigate intracellular sigma peptide (ISP) targeting PTPσ, its effects on autophagy, and synaptic reorganization in SCI. We found that ISP increased the level of autophagosome marker LC3B-II/I and decreased autophagosome degradation marker p62 in SCI, suggesting activated autophagy flux. ISP restored autophagosome-lysosome fusion-related protein syntaxin 17 (STX17) and lysosome-associated membrane protein 2 (LAMP2), indicating activated autophagosome-lysosome fusion. ISP increased pre-synaptic marker synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) expression and improved excitatory synapse marker vesicular glutamate transporter 1 (VGLUT1) and SYN in SCI, suggesting improved synaptic reorganization. ISP promoted axon marker neurofilament and growth-related GAP-43 expression in SCI. ISP rescued a preserved number of motor neurons and improved neurobehavioral recovery after SCI. Our study extended the CSPG-PTPσ inhibition role in activating autophagy flux, axon and synaptic reorganization, and functional recovery in SCI.

Magnesium Ascorbyl Phosphate Promotes Bone Formation Via CaMKII Signaling.

Dysregulation of bone homeostasis is closely related to the pathogenesis of osteoporosis. Suppressing bone resorption by osteoclasts to attenuate bone loss has been widely investigated, but far less effort has been poured toward promoting bone formation by osteoblasts. Here, we aimed to explore magnesium ascorbyl phosphate (MAP), a hydrophilic and stable ascorbic acid derivative, as a potential treatment option for bone loss disorder by boosting osteoblastogenesis and bone formation. We found that MAP could promote the proliferation and osteoblastic differentiation of human skeletal stem and progenitor cells (SSPCs) in vitro. Moreover, MAP supplementation by gavage could alleviate bone loss and accelerate bone defect healing through promoting bone formation. Mechanistically, we identified calcium/calmodulin-dependent serine/threonine kinase IIα (CaMKIIα) as the target of MAP, which was found to be directly bound and activated by MAP, then with a concomitant activation in the phosphorylation of ERK1/2 (extracellular regulated kinase 1/2) and CREB (cAMP-response element binding protein) as well as an elevation of C-FOS expression. Further, blocking CaMKII signaling notably abolished these effects of MAP on SSPCs and bone remodeling. Taken together, our data indicated that MAP played an important role in enhancing bone formation through the activation of CaMKII/ERK1/2/CREB/C-FOS signaling pathway and may be used as a novel therapeutic option for bone loss disorders such as osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

The Natural Product Andrographolide Ameliorates Calcific Aortic Valve Disease by Regulating the Proliferation of Valve Interstitial Cells via the MAPK-ERK Pathway.

Calcific aortic valve disease (CAVD) is an active pathobiological process that involves fibrosis and calcification of aortic valve leaflets, thereby causing cardiac hemodynamic changes and eventually heart failure. Cell proliferation changes at the initial stage of CAVD are an important target for pharmaceutical intervention. This study aimed to investigate whether andrographolide (AGP) could inhibit the proliferation of valve interstitial cells (VICs) in vitro and in vivo to delay the process of CAVD. Cell proliferative factors were tested in both healthy and CAVD aortic valve samples. Cell cycle, cell growth, and calcification of VICs were assessed using flow cytometry, CCK8 assay, EdU staining, and Alizarin Red S staining. The expression of cell proliferative factors and osteogenic factors were quantified by qRT-PCR or immunofluorescence staining. The interaction between AGP and ERK (extracellular regulated protein kinases) was detected by molecular docking. In addition, a high-fat diet-fed animal model was used to verify the effect of AGP on CAVD in vivo. In conclusion, we found that AGP ameliorates aortic valve incrassation by inhibiting cell proliferation via the MAPK-ERK signaling pathway. Therefore, AGP is a promising drug that prevents the occurrence of CAVD via regulating cell proliferation.

Substrate stiffness regulates differentiation of induced pluripotent stem cells into heart valve endothelial cells.

Substrate stiffness has been indicated as a primary determinant for stem cell fate, being capable of influencing motility, proliferation, and differentiation. Although the effects of stiffness on cardiac differentiation of human-induced pluripotent stem cells (h-iPSCs) have been reported, whether stiffness of polydimethylsiloxane-based substrates could enhance differentiation of h-iPSCs toward heart valve endothelial cells lineage (VECs) or not remains unknown. Herein, we modulated the substrate stiffness to evaluate its effect on the differentiation of h-iPSCs into valve endothelial-like cells (h-iVECs) in vitro and determine the suitable stiffness. The results revealed that VECs-related genes (PECAM1, CDH5, NFATC1, etc.) were significantly increased in h-iVECs obtained from the three substrates compared with h-iPSCs. Gene expression levels and differentiation efficiency were higher in the medium group than in the stiff and soft groups. An increase in substrate stiffness to 2.8 GPa decreased the efficiency of h-iPSCs differentiation into h-iVECs and downregulated VECs specific genes. Through mRNA sequencing, we determined the key genetic markers involved in stiffness guiding the differentiation of cardiac progenitor cells into h-iVECs. Unsupervised hierarchical clustering showed that medium stiffness were more suitable for the differentiation of h-iPSCs into h-iVECs in vitro. Moreover, this process is regulated by the WNT/Calcineurin signaling pathway. Overall, this study demonstrates how stiffness can be used to enhance the h-iVECs differentiation of iPSCs and emphasizes the importance of using substrate stiffness to accomplish a more specific and mature differentiation of h-iVECs for future therapeutic and tissue engineering valve applications. STATEMENT OF SIGNIFICANCE: Several studies have examined the stiffness-induced cell fate from pluripotent stem cells during the stage of mesoderm cell differentiation. This is the first research that rigorously examines the effect of substrate stiffness on human valve endothelial-like cells differentiation from cardiac progenitor cells. We found that the medium stiffness can increase the differentiation efficiency of h-iVECs from 40% to about 60%, and this process was regulated by the WNT/CaN signaling pathway through the activation of WNT5a. Substrate stiffness not only increases the differentiation efficiency of h-iVECs, but also improves its cellular functions such as low-density lipoprotein uptake and NO release. This study emphasizes the importance of using substrate stiffness to accomplish a more specific and mature differentiation of h-iVECs.

Optimized Langendorff perfusion system for cardiomyocyte isolation in adult mouse heart.

With the rapid development of single-cell sequencing technology, the Langendorff perfusion system has emerged as a common approach to decompose cardiac tissue and obtain living cardiomyocytes to study cardiovascular disease with the mechanism of cardiomyocyte biology. However, the traditional Langendorff perfusion system is difficult to master, and further, the viability and purity of cardiomyocytes are frequently unable to meet sequencing requirements due to complicated devices and manipulate processes. Here, we provide an optimized Langendorff perfusion system with a simplified and standardized operating protocol which utilizes gravity as the perfusion pressure, includes a novel method for bubbles removing and standardizes the criteria for termination of digestion. We obtained stable cardiomyocyte with high viability and purity after multiple natural gravity sedimentation. The combination of the optimized Langendorff perfusion system and the multiple natural gravity sedimentation provides a stable system for isolating adult mouse heart, which will provide higher-quality cardiomyocytes for further experiments.

A riboflavin-ultraviolet light A-crosslinked decellularized heart valve for improved biomechanical properties, stability, and biocompatibility.

Tissue-engineered heart valves are a promising alternative to current valve substitutes. As the main scaffold of tissue-engineered heart valves, the decellularized heart valve (DHV) has problems such as biomechanical property damage and rapid degradation. In this study, we applied a photo-crosslinking reaction induced by riboflavin and ultraviolet light A (UVA) in the DHV for improving its biomechanical properties and stability. The results showed that the biomechanical properties of the DHV significantly improved following riboflavin-UVA (R-UVA) crosslinking. Moreover, the R-UVA-crosslinked DHV (R-UV-DHV) showed better resistance to enzymatic degradation in vitro, with significantly higher thermal denaturation temperature compared to that of the untreated DHV, indicating that the stability of the R-UV-DHV improved. Histological staining and scanning electron microscopy showed that the leaflet ultrastructure was preserved better after R-UVA crosslinking compared to a glutaraldehyde-crosslinked DHV. In addition, we found that the R-UV-DHV exhibited excellent human umbilical vein endothelial cell adhesion and cells could readily grow on its surface. In an in vitro anti-calcification experiment, the R-UV-DHV demonstrated non-calcifying properties in a simulated body fluid. Furthermore, the R-UV-DHV showed characteristics of slow degradation, non-calcification, and reduced pro-inflammatory response through a rat subcutaneous implantation model. As a result, R-UVA can effectively crosslink the DHV and the R-UV-DHV possessed satisfactory biocompatibility. R-UVA crosslinking can be a new approach for improving the performance of the DHV to prepare a better scaffold for tissue-engineered valves.

Untangling the co-effects of oriented nanotopography and sustained anticoagulation in a biomimetic intima on neovessel remodeling.

Constructing a small-diameter artificial blood vessel with biological functions and mechanical compliance comparable to native tissues is still a major challenge in vascular tissue engineering. To address the issues of severe thrombosis and unsatisfactory long-term patency in small-diameter vascular grafts, herein we designed a specifically biomimetic intima with an oriented nanotopographical structure and covalently immobilized anticoagulant molecules. The mixture of heparinized silk fibroin (SF-Hep) and polycaprolactone (PCL) was used to produce oriented inner layer and pure PCL was used to fabricate vertically porous outer layer by a two-step cross-electrospinning. Our findings showed that the immobilized heparin significantly influenced adherence and activation of platelets while the oriented nanotopography mainly manipulated the elongation and aligned growth of endothelial cells as well as hemodynamics of blood flow. More importantly, two factors of the oriented structure and anticoagulation presented the obviously synergistic effects on rapid endothelialization, long-term patency and remodeling of neovessel. Consequently, the current study successfully combined biochemical induction of heparin molecule and biophysical stimulation of oriented nanotopography to create an off-the-shelf small-diameter vascular graft with excellent antithrombosis in the early stage and long-term patency in the late stage.

Remodeling of a Cell-Free Vascular Graft with Nanolamellar Intima into a Neovessel.

Advances in cardiovascular materials have brought us improved artificial vessels with larger diameters for reducing adverse responses that drive acute thrombosis and the associated complications. Nonetheless, the challenge is still considerable when applying these materials in small-diameter blood vessels. Here we report the biomimetic design of an acellular small-diameter vascular graft with specifically lamellar nanotopography on the luminal surface via a modified freeze-cast technique. The experimental findings verify that the well-designed nanolamellar structure is able to inhibit the adherence and activation of platelets, induce oriented growth of endothelial cells, and eventually remodel a neovessel to maintain long-term patency in vivo. Furthermore, the results of numerical simulations in physically mimetic conditions reveal that the regularly lamellar nanopattern can manipulate blood flow to reduce the flow disturbance compared with random topography. Our current work not only creates a freeze-cast small-diameter vascular graft that employs topographic architecture to direct the vascular cell fates for revasculature but also rekindles confidence in biophysical cues for modulating in situ tissue regeneration.

Clinical features of acute human immunodeficiency virus infection in Taiwan: A multicenter study.

BACKGROUND/PURPOSE: Acute HIV infection is characterized by a high concentration of HIV RNA in the plasma and rapid depletion of the CD4 cell count. This multicenter, retrospective observational study aimed to characterize the manifestations of acuteHIV infection in Taiwan. METHODS: Between 1 January 2012 and 31 December 2016, all patients aged 20 years or greater who presented with acute HIV infection were included. Demographic and clinical characteristics of the patients at diagnosis were collected. Baseline laboratory assessment included hemogram, CD4 count, plasma HIV RNA load (PVL), serologic markers of syphilis and hepatitis A, B, and C viruses, and serum biochemistry. RESULTS: The proportion of acute HIV infection was 6.9% among the patients with newly diagnosed HIV infection during the study period. The most common presenting symptoms of acute HIV infection were fever, fatigue, and myalgia. The median PVL at diagnosis was 5.9 log10 copies/ml, and median CD4 count was 307 cells/mm3. A total of 68 patients (27%) had baseline CD4 count less than 200 cells/mm3. Multiple logistic regression analysis, showed that the baseline CD4 count (OR, 4.02; p = 0.013) and aspartate aminotransaminase levels (OR, 3.49; p = 0.002) were associated with high PVL (>5 log10 copies/ml); and high baseline PVL (OR, 2.64; p = 0.002) was associated with symptomatic acute HIV infection. CONCLUSIONS: Manifestations of acute HIV infection are nonspecific and of wide spectrum ranging from fever to severe illness. A higher proportion of patients with initial CD4 counts of 200 cells/mm3 or less during acute HIV infection warrants early, timely diagnosis and treatment to prevent rapid disease progression.