Piezo1 channel-mediated Ca2+ signaling inhibits lipopolysaccharide-induced activation of the NF-κB inflammatory signaling pathway and generation of TNF-α and IL-6 in microglial cells.

Microglial cells are crucial in maintaining central nervous system (CNS) homeostasis and mediating CNS disease pathogenesis. Increasing evidence supports that alterations in the mechanical properties of CNS microenvironments influence glial cell phenotypes, but the mechanisms regulating microglial cell function remain elusive. Here, we examined the mechanosensitive Piezo1 channel in microglial cells, particularly, how Piezo1 channel activation regulates pro-inflammatory activation and production of pro-inflammatory cytokines, using BV2 and primary microglial cells. Piezo1 expression in microglial cells was detected both at mRNA and protein levels. Application of Piezo1 channel activator Yoda1 induced Ca2+ flux to increase intracellular Ca2+ concentration that was reduced by treatment with ruthenium red, a Piezo1 inhibitor, or Piezo1-specific siRNA, supporting that Piezo1 functions as a cell surface Ca2+ -permeable channel. Priming with lipopolysaccharide (LPS) induced microglial cell activation and production of TNF-α and IL-6, which were inhibited by treatment with Yoda1. Furthermore, LPS priming induced the activation of ERK, p38 MAPKs, and NF-κB. LPS-induced activation of NF-κB, but not ERK and p38, was inhibited by treatment with Yoda1. Yoda1-induced inhibition was blunted by siRNA-mediated depletion of Piezo1 expression and, furthermore, treatment with BAPTA-AM to prevent intracellular Ca2+ increase. Collectively, our results support that Piezo1 channel activation downregulates the pro-inflammatory function of microglial cells, especially production of TNF-α and IL-6, by initiating intracellular Ca2+ signaling to inhibit the NF-κB inflammatory signaling pathway. These findings reveal Piezo1 channel activation as a previously unrecognized mechanism regulating microglial cell function, raising an interesting perspective on targeting this molecular mechanism to alleviate neuroinflammation and associated CNS pathologies.

Risk factors of cardiovascular and cerebrovascular diseases in young and middle-aged adults: A meta-analysis.

BACKGROUND: The risk factors for cardiovascular and cerebrovascular diseases in young and middle-aged people have not yet been determined. We conducted a meta-analysis to find the risk factors for cardiovascular and cerebrovascular diseases, in order to provide guidance for the prevention of diseases in the young and middle-aged population. METHODS: We searched PubMed, Embase, Cochrane Library from the establishment of the database to Mar 2022. We included case-control or cohort studies reporting risk factors for cardiovascular and cerebrovascular disease in young and middle-aged adults. We excluded repeated publication, research without full text, incomplete information or inability to conduct data extraction and animal experiments, reviews and systematic reviews. STATA 15.1 was used to analyze the data. RESULTS: The pooled results indicated that increased systolic blood pressure was significantly associated with increased risk of any stroke, ischemic stroke and hemorrhagic stroke. Body Mass Index (BMI), current smoking, hypertension, and diabetes were significantly associated with increased risk of any stroke and ischemic stroke. Atrial fibrillation was only significantly associated with increased risk of any stroke. Increased total cholesterol was significantly associated with an increased risk of ischemic stroke, whereas increased triglycerides were significantly associated with a decreased risk of ischemic stroke. In addition, increased hypertension was also significantly associated with an increased risk of acute coronary syndrome. CONCLUSION: Our pooled results show that BMI, current smoking, atrial fibrillation, hypertension, systolic blood pressure, and total cholesterol can be used as risk factors for cardiovascular and cerebrovascular diseases in young people, while triglycerides can be used as protective factors for cardiovascular and cerebrovascular diseases in young and middle-aged adults.

NEDD4L inhibits cell viability, cell cycle progression, and glutamine metabolism in esophageal squamous cell carcinoma via ubiquitination of c-Myc.

Esophageal squamous cell carcinoma (ESCC) is a common subtype of esophageal cancer with high incidence. Surgery remains the main strategy for treatment of ESCC at early stage. However, the treatment outcome is unsatisfactory. Therefore, finding new therapeutics is of great importance. In the present study, we measured the level of NEDD4L, an ubiquitin protein ligase, in clinical samples and investigated the effects of NEDD4L on cell viability, cell cycle progression, and glutamine metabolism in TE14 cells determined by CCK-8 assay, flow cytometry and biochemical analysis, respectively. The results show that NEDD4L is significantly decreased in ESCC specimens, and its decreased expression is associated with a poor clinical outcome. Overexpression of NEDD4L significantly inhibits cell viability, cell cycle progression, and glutamine metabolism in TE14 cells. Mechanistic study indicates that NEDD4L regulates tumor progression through ubiquitination of c-Myc and modulation of glutamine metabolism. NEDD4L inhibits cell viability, cell cycle progression, and glutamine metabolism in ESCC by ubiquitination of c-Myc to decrease the expressions of GLS1 and SLC1A5. Our findings highlight the importance of NEDD4L/c-Myc signaling in ESCC.

Role of endoplasmic reticulum stress in apoptosis induced by HK2 inhibitor and its potential as a new drug combination strategy.

Compared with normal cells, tumor cells mainly obtain energy through aerobic glycolysis. Hexokinase 2 (HK2) plays a key role in the regulation of tumor cell aerobic glycolysis, and targeting HK2 has become a new strategy for cancer treatment. However, little is known about the role of HK2 in colon cancer and the regulation of its targeted inhibitors. In this study, we found that the expression of HK2 in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the expression level of HK2 in metastatic colorectal cancer was further increased. Meanwhile, the expression level of HK2 was closely related to clinical TNM stage and outcome of colorectal cancer patients. We provide here evidence that HK2 inhibitor 3-Bromopyruvate acid (3-BP) can significantly inhibit the survival and proliferation of colon cancer cells, and induce apoptosis through mitochondrial apoptosis signaling pathway. In addition, we found that 3-BP can also induce endoplasmic reticulum stress in colon cancer cells, the mechanism may be through the increase of intracellular calcium concentration. In vitro and in vivo experiments showed that inhibition of endoplasmic reticulum stress could further increase the proliferation inhibition and apoptosis induced by 3-BP. Collectively, our results show that HK2 is highly expressed in colorectal cancer. 3-BP, an inhibitor of HK2, can induce apoptosis and endoplasmic reticulum stress in colon cancer cells. Endoplasmic reticulum stress plays a protective role in cell death induced by 3-BP. This result suggested that targeting HK2 and endoplasmic reticulum stress may be a valuable strategy in targeted and combination therapy of colon cancer.

A critical role of the KCa 3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells.

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca2+ -activated K+ channel, KCa 3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the KCa 3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the KCa 3.1 channel expression and pharmacological or genetic inhibition of the KCa 3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the KCa 3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.

Design, synthesis and biological evaluation of bromophenol-thiazolylhydrazone hybrids inhibiting the interaction of translation initiation factors eIF4E/eIF4G as multifunctional agents for cancer treatment.

The eukaryotic initiation factor 4E (eIF4E) is an emerging anticancer drug target for specific anticancer therapy as a promising approach to overcome drug resistance and promote chemotherapy antitumor efficacy. A series of bromophenol-thiazolylhydrazone hybrids were designed, synthesized and evaluated for their antitumor activities. Among of them, the most potent compound 3e (EGPI-1) could inhibit the eIF4E/eIF4G interaction. Further mechanism study demonstrated EGPI-1 played an antitumor role in multiple modes of action including regulating the activity of eIF4E by inhibiting the phosphorylation of eIF4E and 4EBP1, disrupting mitochondrial function through the mTOR/4EBP1 signaling pathway, and inducing autophagy, apoptosis and ROS generation. Moreover, EGPI-1 showed good safety and favorable pharmacokinetic properties in vivo. These observations demonstrate that EGPI-1 may serve as an excellent lead compound for the development of new anticancer drugs that target the eIF4E/eIF4G interface and as a chemical genetic probe to investigate the role of the eIF4E in biological processes and human diseases.

[Efficacy of Bortezomib for Maintenance therapy of Patients with Multiple Myeloma].

OBJECTIVE: To observe the efficacy of chemotherapy consisted of bortezomib as main druy in maintenance therapy for recurrence of newly diagnosed MM patients. METHODS: The clinical data and outcome of 37 MM patients during 2008-2013 were analyzed retrospectively, the 37 MM patients were divided into 2 group: 19 cases including 13 cases of newly diagnosed MM with symptoms and 6 cases of relapsed refractory MM were enrolled in group A; 17 cases of newly diagnosed MM with symptoms were enrolled in group B. The patients of group A received maintenance therapy consisted of bortezomib plus dexamethasone (VD group), while the patient group B received maintenance therapy consisted of melphalan plus prednisone(MP group), then the therapeutic efficacy of 2 group was compared. RESULTS: The overall response rate(ORR) in VD groupe was 84.2%(16/19), out of which CR rate reached 42%(8/19), PR rate reached 31.6%(6/19), MR rate reached 10.5%(3/19). During median follow-up for 21.8(5-51) months, death occurred, while the ORR in MP group was 52(9/17), out of which CR rate was 23.5%(4/17), PR rate reached 23.5%(4/17), MR rate reached 5.9%(1/17). Druing median follow-up for 16.4(4-39) months, the worteity reaced 64.7%(11/17). The differencr between 2 groups was significant(P<0.05). The median OS time of patients in VD group was 21.6 months, that in MP group was 17.9 months(P<0.05). The median PFS in VD group and MP group were 13.4 and 9.4 months respectively(P<0.001). CONCLUSION: The ORR and CR rates of bortezomib maintenance therapy for newly diagnosed and relapsed / refractory MM patients are very high, and its toxicity can be controlled, therefore, the patients need maintenance therapy after remission.

Electrospun PELCL membranes loaded with QK peptide for enhancement of vascular endothelial cell growth.

One of the major challenges in tissue engineering of small-diameter vascular grafts is to inhibit intimal hyperplasia and keep long-term patency after implantation. Rapid endothelialization of the grafts could be an effective approach. In this study, QK, a peptide mimicking vascular endothelial growth factor, was selected as the bioactive substrate and loaded in electrospun membranes for enhancement of vascular endothelial cell growth. In detail, QK peptide was firstly introduced with poly(ethylene glycol) diacrylate into a thiolated chitosan solution that could transfer into hydrogel. Then, suspensions or emulsions of poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) (PELCL) containing QK peptide (with or without chitosan hydrogel) were electrospun into fibrous membranes. For comparison, the electrospun PELCL membrane without QK was also fabricated. Results of release behaviors showed that the electrospun membranes, especially that contained chitosan hydrogel prepared by suspension electrospinning, could successfully encapsulate QK peptide and maintain its secondary structure after released. In vitro cell culture studies exhibited that the release of QK peptide could accelerate the proliferation of vascular endothelial cells in the 9 days. It was suggested that the electrospun PELCL membranes loaded with QK peptide might have potential applications in vascular tissue engineering.

Targeted delivery of microRNA-126 to vascular endothelial cells via REDV peptide modified PEG-trimethyl chitosan.

Manipulation of gene expression by means of microRNAs (miRNAs) is one of the emerging strategies to treat cardiovascular and cancer diseases. Nevertheless, efficient delivery of miRNAs to a specific vascular tissue is limited. In this work, a short peptide Arg-Glu-Asp-Val (REDV) was linked to trimethyl chitosan (TMC) via a bifunctional poly(ethylene glycol) (PEG) linker for the targeted delivery of microRNA-126 (miRNA-126) to vascular endothelial cells (VECs). The morphology, serum stability and cytotoxicity of the polyplex/miRNA complexes, namely, TMC/miRNA, TMC-g-PEG/miRNA and TMC-g-PEG-REDV/miRNA, were investigated along with the cellular uptake, proliferation and in vitro miRNA transfection efficiency. By REDV modification, the TMC-g-PEG-REDV/miRNA complex showed negligible cytotoxicity, increased expression of miRNA-126 and enhanced VEC proliferation compared with the TMC/miRNA and TMC-g-PEG/miRNA complexes. In particular, the approaches adopted for the miRNA delivery and targeted peptide REDV modification promote the selective uptake and the growth of VECs over vascular smooth muscle cells. It was suggested that the REDV peptide-modified TMC-g-PEG polyplex could be potentially used as a miRNA carrier in artificial blood vessels for rapid endothelialization.

Microencapsulation of mechano growth factor E peptide for sustained delivery and bioactivity maintenance.

Mechano growth factor (MGF) and its C-terminal E-peptide with 24 amino acids, MGF-Ct24E, have superiority in resolving the delayed or failed bone repair derived from shortness of suitable biomechanical stimulation. The chitosan/tripolyphosphate microspheres encapsulated with MGF-Ct24E (CS/TPP/MGF-Ct24E) are prepared using emulsion-ionic cross-linking method in order to achieve the sustained release and preserve the bioactivity of MGF-Ct24E. The microspheres are micron-sized and spherical in shape with smooth surface morphology. The TPP component disintegrates in advance of CS matrix and the MGF-Ct24E maintains sustained delivery during in vitro hydrolytic degradation. With the disappearance of TPP, the total weight loss of CS/TPP/MGF-Ct24E is 32% and the release amount of MGF-Ct24E reaches 84.6% after degrading for 2 weeks. In vitro bioactivity assays reveal that the MGF-Ct24E can accelerate MC3T3-E1 cells proliferation and delay their differentiation as well. The encapsulated MGF-Ct24E shows long-term effects after being loaded in the CS/TPP microspheres and the cells exhibit excellent morphology on the surface of microspheres. The continuous delivery of MGF-Ct24E provides a new perspective on resolving the unsatisfactory bone reconstruction associated with microgravity and stress shielding.

Increased proliferation and differentiation of pre-osteoblasts MC3T3-E1 cells on nanostructured polypyrrole membrane under combined electrical and mechanical stimulation.

Polypyrrole (PPy), as an electrical conductive polymer, has been widely investigated in biomedical fields. In this study, PPy membrane at nanoscale was electrically deposited on indium-tin oxide glass slide with sodium p-toluenesulfonate as supporting electrolyte. Electropolymerization of PPy was performed under a constant 800 mV voltage for 10 seconds. Chemical compositions and morphology were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The results showed that the nanoscaled PPy particles distributed uniformly and the average diameter of PPy particles was 62 nm. Since bone cells can respond to both electrical and mechanical stimulation in vivo, pre-osteoblasts MC3T3-E1 cells were cultured ort nanostructured PPy membrane under the combined electrical and mechanical stimulation. The nano-PPy membrane was conducive to transferring uniform electrical stimulation and applying steady mechanical stimulation. It is suggested that the combined stimulation did not affect cells morphologies significantly. However, cell proliferation tested by MTT, alkaline phosphatase activities, and gene expression of Collagen-I indicated that combined stimulation can enhance the proliferation and differentiation of MC3T3-E1 cells more efficiently than single electrical stimulation or single mechanical stimulation. The combined stimulation through a nano-PPy membrane may provide a highly potential stimulated method in bone tissue engineering.

Performance of a multilayered small-diameter vascular scaffold dual-loaded with VEGF and PDGF.

The urgent needs of functional arterial replacements for curing the vascular system diseases have been proposed for many years. However, an ideal small-diameter vascular scaffold, which is nonthrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of natural vessels, and supports neovascular tissue reconstruction, is still in progress. For this purpose, we previously attempted dual-delivery of VEGF and PDGF by double-layered electrospun membranes. Here, a multilayered vascular scaffold in 1.5-mm diameter with sufficient mechanical properties was developed by electrospinning from poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) (PELCL), poly(L-lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL) and gelatin. Spatio-temporal releases of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGF) were specially controlled by the inner PELCL and middle PLGA layers, respectively, and the outer PCL layer contributed to the mechanical stability. Introduction of gelatin improved vascular endothelial cells adhesion at first, and loosen membrane after its degradation facilitated vascular smooth muscle cells (VSMCs) ingrowth. Cell activities indicated dual release of growth factors promoted endothelialization and inhibited VSMCs hyperproliferation. The small-diameter vascular scaffold dual-loading VEGF and PDGF could maintain patency in rabbit left common carotid artery for 8 weeks. It is concluded that the specially prepared fibrous scaffold in multilayer could benefit blood vessel reconstruction.

Galanin protects against nerve injury after shear stress in primary cultured rat cortical neurons.

The neuropeptide galanin and its receptors (GalR) are found to be up-regulated in brains suffering from nerve injury, but the specific role played by galanin remains unclear. This study aimed to explore the neuroprotective role of galanin after shear stress induced nerve injury in the primary cultured cortical neurons of rats. Our results demonstrated that no significant changes in cell death and viability were found after galanin treatment when subjected to a shear stress of 5 dyn/cm(2) for 12 h, after increasing magnitude of shear stress to 10 dyn/cm(2) for 12 h, cell death was significantly increased, while galanin can inhibit the nerve injury induced by shear stress with 10 dyn/cm(2) for 12 h. Moreover, Gal2-11 (an agonist of GalR2/3) could also effectively inhibit shear stress-induced nerve injury of primary cultured cortical neurons in rats. Although GalR2 is involved in the galanin protection mechanism, there was no GalR3 expression in this system. Moreover, galanin increased the excitatory postsynaptic currents (EPSCs), which can effectively inhibit the physiological effects of shear stress. Galanin was also found to inhibit the activation of p53 and Bax, and further reversed the down regulation of Bcl-2 induced by shear stress. Our results strongly demonstrated that galanin plays a neuroprotective role in injured cortical neurons of rats.

Dual-delivery of VEGF and PDGF by double-layered electrospun membranes for blood vessel regeneration.

Tissue engineering of small-diameter blood vessels is still challenging because of restenosis and burst. To prevent thrombosis, rapid endothelialization along the lumen of grafts is intended, followed by proliferation of vascular smooth muscle cells (VSMCs) around the exterior for compliance. To this goal, two modified coaxial electrospinning techniques were developed to encapsulate vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGF), respectively, to regulate proliferation of vascular endothelial cells (VECs) and VSMCs. Release profiles, in vitro cell proliferation and in vivo implantation of double-layered electrospun membranes were investigated, and what made it special was the electrospun membranes were composed of chitosan hydrogel/poly(ethylene glycol)-b-poly(L-lactide-co-caprolactone) (PELCL) electrospun membrane loaded with VEGF as the inner layer and emulsion/PELCL electrospun membrane-loaded PDGF as the outer. It was found that dual-release of VEGF and PDGF could accelerate VEC proliferation in the first 6 days, and modulate slow VSMC proliferation in the initial 3 days whereas generate rapid proliferation after day 6, which is of great benefit to blood vessel regeneration. Four weeks of in vivo replacement of rabbit carotid artery demonstrated that VECs and VSMCs developed on the lumen and exterior of vascular grafts, respectively, and no thrombus or burst appeared. It was concluded that dual-delivery of VEGF and PDGF by the modified electrospun membranes could facilitate revascularization.

Effect of cyclic strain on cardiomyogenic differentiation of rat bone marrow derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Sustained release of VEGF by coaxial electrospun dextran/PLGA fibrous membranes in vascular tissue engineering.

VEGF-loaded core/shell fibrous membranes were prepared by coaxial electrospinning with dextran (DEX) as the core component and poly(lactide-co-glycolide) (PLGA) as the shell polymer, respectively. The electrospun DEX/PLGA fibers were observed by scanning electron microscopy, transmission electron microscopy and confocal microscopy to identify the core/shell fiber structure and the protein distribution. The results of tensile tests showed that the DEX/PLGA membranes possessed lower tensile strength and higher Young's modulus than PLGA one. The release profiles demonstrated that vascular endothelial growth factor (VEGF) release sustained for more than 28 days. Studies on cell viability and spreading demonstrated that the DEX(VEGF)/PLGA membranes positively promoted cell proliferation and cell-membrane interaction, which further testified that the processed VEGF remained bioactivities. Furthermore, the detections for the up-regulation of intercellular adhesion molecular-1 and the release of von Willebrand factor under pathological stimuli, which are related to inflammation process and thrombus formation, exhibited a normal immune response for the DEX(VEGF)/PLGA membrane. These data suggested that the VEGF-loaded fibers could be feasible in vascular tissue engineering.

Effect of fluid shear stress on cardiomyogenic differentiation of rat bone marrow mesenchymal stem cells.

BACKGROUND AND AIMS: Bone marrow mesenchymal stem cells (BMSCs) are a potential source of material for the construction of tissue-engineered cardiac grafts because of their potential to transdifferentiate into cardiomyocytes after chemical treatment or co-culture with cardiomyocytes. Recent evidence has shown that mechanical loads could regulate the BMSC differentiation into osteoblasts and endothelial cells through various signaling pathways. We investigated whether fluid shear stress (FSS), which is a mechanical load generated by fluid flow, can regulate rat BMSC (rBMSC) differentiation into cardiomyocytes. METHODS: rBMSCs were isolated from marrow of rat femur and tibia using density gradient centrifugation combined with adhesion method and identified with surface marker, proliferation character and differentiation potential in vitro. Cultured rBMSCs with or without 5-azacytidine (5-aza) treatment were exposed to laminar shear stress with a parallel plate-type device and analyzed by RT-PCR, immunocytochemistry, FACS and Western-blotting for the cardiomyogenic differentiation. RESULTS: Appropriate FSS treatment alone induced cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, when rBMSC cultures were exposed to both FSS and 5-aza, expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. CONCLUSIONS: The results demonstrate that FSS is an important factor affecting cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations.

Bone marrow mesenchymal stem cells (MSCs) have multi-differentiation capability. Their endothelial cell (EC) oriented differentiation is the key to vasculogenesis, in which both mechanical and chemical stimulations play important roles. Most previous studies reported individual effects of VEGF or fluid shear stress (SS), when MSCs were subjected to shear stress of 10-15 dyn/cm(2) over 24hr. In this paper, we investigated responses of MSCs from young Sprague Dawley rats to shear stress, VEGF and the combination of the two stimuli. Our study showed that the combined stimulation of shear stress and VEGF resulted in more profound EC oriented differentiation of MSCs in comparison to any individual stimulation. Furthermore, we subjected MSCs to prolonged period of fluid shear stimulation, i.e. 48 hr rather than 24hr, and increased the magnitude of the shear stress from 10 dyn/cm(2) to 15, 20 and 25 dyn/cm(2). We found that without VEGF, the endothelium oriented differentiation of MSCs that was seen following 24hr of shear stimulation was largely abolished if we extended the shear stimulation to 48hr. A similar sharp decrease in MSC differentiation was also observed when the magnitude of the shear stress was increased from 10-15 dyn/cm(2) to 20-25 dyn/cm(2) in 24hr shear stimulation studies. However, with combined VEGF and fluid shear stimulation, most of the endothelial differentiation was retained following an extended period, i.e. at 48 hr, of shear stimulation. Our study demonstrates that chemical and mechanical stimulations work together in determining MSC differentiation dynamics.

Molecular modeling and affinity determination of scFv antibody: proper linker peptide enhances its activity.

One of existing strategies to engineer active antibody is to link V(H) and V(L) domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or single-chain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned V(H) and V(L) domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and approximately 24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions.

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.