Research Progress on the Pathogenesis of Aortic Aneurysm and Dissection in Metabolism.

Aortic aneurysm and dissection are complicated diseases having both high prevalence and mortality. It is usually diagnosed at advanced stages and posing diagnostic and therapeutic challenges due to the limitations of current detecting methods for aortic dissection used in clinics. Metabonomics demonstrated its great potential capability in the early diagnosis and personalized treatment of several diseases. Emerging evidence suggests that metabolic disorders including amino acid metabolism, glycometabolism, and lipid metabolism disturbance are involved in the pathogenesis of aortic aneurysm and dissection by affecting multiple functional aortic cells. The purpose of this review is to provide new insights into the metabolism alterations and their related regulatory mechanisms with a focus on recent advances and findings and provide a theoretical basis for the diagnosis, prevention, and drug development for aortic aneurysm and dissection.

Clinical Outcomes by Consolidation of Bone Marrow Stem Cell Therapy and Coronary Artery Bypass Graft in Patients With Heart Failure With Reduced Ejection Fraction: A Meta-analysis.

Bone marrow stem cell (BMSC) transplantation during coronary artery bypass graft (CABG) is an innovative treatment for ischemic heart disease (IHD). We conduct a meta-analysis to examine whether patients with IHD presenting heart failure with reduced ejection fraction (HFrEF) can be beneficent from CABG with additional BMSC transplantation. Electronic searches were performed on PubMed, EMBASE, Cochrane Library, and ClinicalTrials.gov from their inception to July 2021. The efficacy was based on left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), left ventricular end-diastolic volume (LVEDV), left ventricular end-diastolic volume index (LVEDVi), left ventricular end-systolic volume index (LVESVi), and 6-min walk test (6MWT) change after treatment. Eight randomized-controlled trials (RCTs) were included in this meta-analysis, with a total of 350 patients. Results showed BMSC transplantation significantly improved the LVEF [mean difference (MD) = 6.23%, 95% confidence interval (CI): 3.22%-9.24%, P < 0.0001], LVEDVi (MD = -20.15 ml/m2, 95% CI: -30.49 to -9.82 ml/m2, P < 0.00001), and LVESVi (MD = -17.69 ml/m2, 95% CI: -25.24 to -10.14 ml/m2, P < 0.00001). There was no statistically significant difference in the improvement of LVEDD, LVEDV, and 6MWT between the cell transplantation group and control groups. Subgroup analysis revealed that the intervention for control group could affect the efficacy of BMSC transplantation. Sensitivity analysis found the conclusion of LVEDD, LVEDV, and 6MWT changes was not stable. Therefore, among patients with IHD presenting HFrEF, BMSC transplantation during CABG is promising to be beneficial for postoperative left ventricular (LV) function improvement. However, according to the unstable results of the sensitivity analysis, it cannot be concluded whether the extra step has a positive effect on left ventricular remodeling and exercise capacity. RCTs with larger cohorts and more strict protocols are needed to validate these conclusions.

Serum cystatin C is a potential predictor of short-term mortality and acute kidney injury in acute aortic dissection patients: a retrospective cohort study.

Background: Serum cystatin C concentration is associated with cardiovascular disease. However, the relationship between cystatin C and acute aortic dissection (AAD) remains unclear. In the current study, we aim to evaluate the predictive value of cystatin C in the occurrence of acute kidney injury (AKI) and the prognosis of AAD patients. Methods: The patients with AAD admitted to our hospital from November 2019 through January 2022 were consecutively included in the retrospective cohort study. A complete blood cell count, serum biochemistry tests, including cystatin C and creatinine, in-hospital mortality and the incidence of AKI were recorded. All the patients were categorized into four groups according to the quartile of their serum cystatin C levels. Multivariate logistic and Cox regression analyses were conducted to determine the independent risk factors for the incidence of AKI and the prognosis of AAD patients, respectively. Kaplan-Meier analyses and log-rank tests were used to evaluate differences in survival. Receiver operating characteristic (ROC) curves were used to assess the predictive value of cystatin C for short-term mortality and the incidence of AKI in AAD patients. Results: A total of 357 patients were included in this study. The results showed that the higher the concentration of cystatin C, the higher the level of serum creatinine and the higher the incidence of AKI. Mortality was significantly higher in the group with serum cystatin C levels >1.18 mg/L. Type A AAD, white blood cell count >10×109/L, platelet count <100×109/L, and serum cystatin C concentration >1.18 mg/L [adjusted hazards ratio (HR) =2.405, 95% confidence interval (CI), 1.029-4.063, P=0.041] were independent risk factors for in-hospital mortality. Cystatin C levels >1.18 mg/L remained an independent predictor of AKI in AAD after adjusting for the confounding [odds ratio (OR) 76.489, 95% CI, 25.586-228.660]. The areas under the ROC curves of cystatin C in predicting the mortality and incidence of AKI in AAD patients were 0.655 (95% CI, 0.551-0.760) and 0.807 (95% CI, 0.758-0.856), respectively. Conclusions: In sum, serum cystatin C concentration is a potential predictor of short-term mortality and the incidence of AKI in AAD patients.

Generation of a Human iPSC (ICSSUi002-A) with MTHFR SNP (rs1801133, TT) from Thoracic Aortic Dissection Patient.

Thoracic aortic dissection is a devastating cardiovascular disease with an increasing annual incidence. The homozygous mutation in rs1801133 site has been accepted for decreased enzyme activity of mutant MTHFR protein, contributing to an accumulated homocysteine in blood. Recently, elevated homocysteine level is causally associated with an increased risk of cardiovascular disease. Conversely, the relationship between rs1801133 and thoracic aortic dissection is poorly understood. Here, the generated human induced pluripotent stem cell (iPSC) line provided a novel strategy for investigating the underlying mechanism of MTHFR mutation (rs1801133, TT) and its implication in the pathogenesis of thoracic aortic dissection.

Evaluation of Circulating Endothelial Progenitor Cells in Abdominal Aortic Aneurysms after Endovascular Aneurysm Repair.

Background and Objectives: Circulating endothelial progenitor cells (EPCs) participate in vascular repair and predict cardiovascular outcomes. The aim of this study was to investigate the correlation between EPCs and abdominal aortic aneurysms (AAAs). Methods and Results: Patients (age 67±9.41 years) suffering from AAAs (aortic diameters 58.09±11.24 mm) were prospectively enrolled in this study. All patients received endovascular aneurysm repair (EVAR). Blood samples were taken preoperatively and 14 days after surgery from patients with aortic aneurysms. Samples were also obtained from age-matched control subjects. Circulating EPCs were defined as those cells that were double positive for CD34 and CD309. Rat models of AAA formation were generated by the peri-adventitial elastase application of either saline solution (control; n=10), or porcine pancreatic elastase (PPE; n=14). The aortas were analyzed using an ultrasonic video system and immunohistochemistry. The levels of CD34＋/CD309＋ cells in the peripheral blood mononuclear cell populations were measured by flow cytometry. The baseline numbers of circulating EPCs (CD34＋/CD309＋) in the peripheral blood were significantly smaller in AAA patients compared with control subjects. The number of EPCs doubled by the 14th day after EVAR. A total of 78.57% of rats in the PPE group (11/14) formed AAAs (dilation ratio ＞150%). The numbers of EPCs from defined AAA rats were significantly decreased compared with the control group. Conclusions: EPC levels may be useful for monitoring abdominal aorta aneurysms and rise after EVAR in patients with aortic aneurysms, and might contribute to the rapid endothelialization of vessels.

HIF-1α overexpression in mesenchymal stem cell-derived exosomes mediates cardioprotection in myocardial infarction by enhanced angiogenesis.

BACKGROUND: Myocardial infarction (MI) is a severe disease that often associated with dysfunction of angiogenesis. Cell-based therapies for MI using mesenchymal stem cell (MSC)-derived exosomes have been well studied due to their strong proangiogenic effect. Genetic modification is one of the most common methods to enhance exosome therapy. This study investigated the proangiogenic and cardioprotective effect of exosomes derived from hypoxia-inducible factor 1-alpha (HIF-1α)-modified MSCs. METHODS: Lentivirus containing HIF-1α overexpressing vector was packaged and used to infect MSCs. Exosomes were isolated from MSC-conditioned medium by ultracentrifugation. Human umbilical vein endothelial cells (HUVECs) were treated under hypoxia condition for 48 h co-cultured with PBS, control exosomes, or HIF-1α-overexpressed exosomes, respectively. Then the preconditioned HUVECs were subjected to tube formation assay, Transwell assay, and EdU assay to evaluate the protective effect of exosomes. Meanwhile, mRNA and secretion levels of proangiogenic factors were measured by RT-qPCR and ELISA assays. In vivo assays were conducted using the rat myocardial infarction model. PBS, control exosomes, or HIF-1α-overexpressed exosomes were injected through tail vein after MI surgery. Heart function was assessed by echocardiography at days 3, 14, and 28. At day 7, mRNA and protein expression levels of proangiogenic factors in the peri-infarction area and circulation were evaluated, respectively. At day 28, hearts were collected and subjected to H&E staining, Masson's trichrome staining, and immunofluorescent staining. RESULTS: HIF-1α-overexpressed exosomes rescued the impaired angiogenic ability, migratory function, and proliferation of hypoxia-injured HUVECs. Simultaneously, HIF-1α-overexpressed exosomes preserved heart function by promoting neovessel formation and inhibiting fibrosis in the rat MI model. In addition, both in vitro and in vivo proangiogenic factors mRNA and protein expression levels were elevated after HIF-1α-overexpressed exosome application. CONCLUSION: HIF-1α-overexpressed exosomes could rescue the impaired angiogenic ability, migration, and proliferation of hypoxia-pretreated HUVECs in vitro and mediate cardioprotection by upregulating proangiogenic factors and enhancing neovessel formation.

Targeted inhibition of endothelial calpain delays wound healing by reducing inflammation and angiogenesis.

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are a well-known family of calcium-dependent cysteine proteases that regulate several processes, including cellular adhesion, proliferation, and migration, as well as inflammation and angiogenesis. CAPNS1, the common regulatory subunit of Calpain-1 and 2, is indispensable for catalytic subunit stabilization and activity. Calpain inhibition has been shown to reduce organ damage in various disease models. Here, we report that endothelial calpain-1/2 is crucially involved in skin wound healing. Using a mouse genetic model where Capns1 is deleted only in endothelial cells, we showed that calpain-1/2 disruption is associated with reduced injury-activated inflammation, reduced CD31+ blood vessel density, and delayed wound healing. Moreover, in cultured HUVECs, inhibition of calpain reduced TNF-α-induced proliferation, migration, and tube formation. Deletion of Capns1 was associated with elevated levels of IκB and downregulation of ß-catenin expression in endothelial cells. These observations delineate a novel mechanistic role for calpain in the crosstalk between inflammation and angiogenesis during skin repair.

The protective effects of HMGA2 in the senescence process of bone marrow-derived mesenchymal stromal cells.

Bone marrow-derived mesenchymal stromal cells (MSCs) have been wildly applied to cell-based strategies for tissue engineering and regenerative medicine; however, they have to undergo the senescence process and thus appeared to be less therapeutic effective. HMGA2, a protein belonged to high mobility group A (HMGA) family, exhibits an inverse expression level related to embryonic development and acts as a developmental regulator in stem cell self-renewal progression. Therefore, we performed senescence-associated ß-galactosidase (SA-ß-gal) staining, transwell assay, to examine the changes of MSCs in different stages and then over-expressed HMGA2 in MSCs by lentivirus transfection. We found the percentage of SA-ß-gal staining positive cells in MSCs from 24-month-old Sprague-Dawley (SD) rats (O-MSCs) was significantly higher compared with MSCs from 2-week-old SD rats (Y-MSCs), and the expression levels of P21 and P53, two senescence-related molecules, were also significantly up-regulated in O-MSCs than in Y-MSCs. In contrast, the HMGA2 expression level in O-MSCs was dramatically down-regulated in contrast to Y-MSCs. In additional, the migration ability in O-MSCs was significantly attenuated than in Y-MSCs. After successfully over-expressed HMGA2 in O-MSCs, the percentage of SA-ß-gal staining positive cells and the expression levels of P21 and P53 were reduced, and the migration ability was improved compared with O-MSCs without treatment. Further, mRNA sequencing analysis revealed that overexpression of HMGA2 changed the expression of genes related to cell proliferation and senescence, such as Lyz2, Pf4, Rgs2, and Mstn. Knockdown of Rgs2 in HMGA2 overexpression O-MSCs could antagonize the protective effect of HMGA2 in the senescence process of O-MSCs.

Selective deletion of endothelial cell calpain in mice reduces diabetic cardiomyopathy by improving angiogenesis.

AIMS/HYPOTHESIS: The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy. METHODS: Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed. RESULTS: Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14 ± 423.51 vs 4708.7 ± 417.93 capillary number/high-power field in prediabetes, 2999.36 ± 854.77 vs 4579.22 ± 672.56 capillary number/high-power field in type 2 diabetes and 2364.87 ± 249.57 vs 3014.63 ± 215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of ß-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed ß-catenin protein expression in cultured endothelial cells. Upregulation of ß-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ß-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress. CONCLUSIONS/INTERPRETATION: These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of ß-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.

Enhancement of Immunoregulatory Function of Modified Bone Marrow Mesenchymal Stem Cells by Targeting SOCS1.

OBJECTIVE: The study aim to investigate the role of microRNA-155 (miR-155) on the immunoregulatory function of bone marrow mesenchymal stem cells (MSCs). METHODS: MSCs were isolated from 2-week-old Sprague-Dawley rats and identified by flow cytometry using anti-CD29, anti-CD44, anti-CD34, and anti-CD45 antibodies. MSCs were transfected with miR155-mimics, miR155-inhibitor, and control oligos, respectively, and then cocultured with spleen mononuclear cells (SMCs). The mRNA levels of Th1, Th2, Th17, and Treg cell-specific transcription factors (Tbx21, Gata3, Rorc, and Foxp3, resp.) and the miR-155 target gene SOCS1 were detected by quantitative real-time PCR (qPCR) in SMCs. The proportion of CD4+ FOXP3+ Treg cells was detected by flow cytometry. In addition, the effects of MSCs transfected with miR-155 on the migration of rat SMCs were investigated by transwell chamber. RESULTS: CD29 and CD44 were expressed in MSCs, while CD34 and CD45 were negative. The percentage of CD4+ FOXP3+ Treg cells in the SMC population was significantly higher compared with that noted in SMCs control group (p < 0.001) following 72 hours of coculture with miR155-mimics-transfected SMCs. In contrast, the percentage of CD4+ FOXP3+ Treg cells in the SMCs cocultured with miR155-inhibitor-transfected MSCs was significantly lower compared with that noted in SMCs control group (p < 0.001). MiR155-mimics-transfected MSCs inhibited the expression of Tbx21, Rorc, and SOCS1, while the expression of Gata3 and Foxp3 was increased. In contrast to the downregulation of the aforementioned genes, miR155-inhibitor-transfected MSCs resulted in upregulation of Tbx21, Rorc, and SOCS1 expression levels and inhibition of Gata3 and Foxp3. In the transwell assay, miR155-mimics-transfected MSCs exhibited lower levels of SMCs migration, while the miR155-inhibitor-transfected MSCs demonstrated significantly higher levels of migration, compared with the blank control group (p < 0.01, resp.). CONCLUSION: miR-155 favors the differentiation of T cells into Th2 and Treg cells in MSCs, while it inhibits the differentiation to Th1 and Th17 cells.

Protective action of bone marrow mesenchymal stem cells in immune tolerance of allogeneic heart transplantation by regulating CD45RB+ dendritic cells.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) could exert a potent immunosuppressive effect and therefore may have a therapeutic potential in T-cell-dependent pathologies. We aimed to examine the effects of BMSCs on immune tolerance of allogeneic heart transplantation and the involvement of CD45RB+ dendritic cells (DCs). METHODS: Bone marrow-derived DCs and BMSCs were co-cultured, with CD45RB expression on the surface of DCs measured by flow cytometry. qRT-PCR and Western blotting were used to detect mRNA and protein levels. Cytometric bead array was performed to determine the serum level of IL-10. Survival time of transplanted heart and expression of CD4+ , CD8+ , IL-2, IL-4, IL-10, IFN-γ were determined. Immunofluorescence assay was employed to determine intensity of C3d and C4d. RESULTS: DCs co-cultured with BMSCs showed increased CD45RB and Foxp3 levels. CD45RB+ DCs co-cultured with T-cells CD4+ displayed increased T-cell CD4+ Foxp3 ratio and IL-10 than DCs. Both of them extended survival time of transplanted heart, decreased histopathological classification and score, intensity of C3d, C4d, proportion of CD4+ , expression levels of IL-2 and IFN-γ, and increased the CD4+ Foxp3 ratio and levels of IL-4 and IL-10. CD45RB+ DCs achieved better protective effects than DCs. CONCLUSION: BMSCs increased the expression of CD45RB in the bone marrow-derived DCs, thereby strengthening immunosuppression capacity of T cells and immune tolerance of allogeneic heart transplantation.

Effect of advanced glycation end products, extracellular matrix metalloproteinase inducer and matrix metalloproteinases on type-I collagen metabolism.

The aim of the study was to examine the association among advanced glycation end products (AGEs), extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMPs), and investigate whether AGEs affect type I collagen (COL-I) through EMMPRIN or MMPs. A co-culture system with the osteoblast-like cells (MC3T3E1) and mouse RAW264.7 cells was employed to examine the effects of AGE-bovine serum albumin (BSA) (50 mg/l), EMMPRIN antibody (5 mg/l) and AGE-BSA+EMMPRIN antibody separately on COL-I expression for 24 h. Culture media were analyzed for the content of COL-I by ELISA. The effect of different concentrations of AGE-BSA (0, 50, 100, 200 and 400 mg/l) for 24 h was assessed on COL-I levels. Finally, semiquantitative RT-PCR was used to detect the osteoblast COL-I mRNA expression and MMP-2 and MMP-9's PMAO were also measured in the culture medium. COL-I content in the culture medium decreased significantly following treatment with AGE-BSA (P<0.05). EMMPRIN antibody increased COL-I content (P<0.05). EMMPRIN antibody+AGE-BSA increased COL-I significantly (P<0.05). Different concentrations of AGE-BSA increased COL-I mRNA expression significantly compared with the control group (P<0.05), and were enhanced with increasing AGE-BSA concentration (P<0.05). Also MMP-2 and MMP-9 secretion increased significantly (P<0.05), with the increasing AGE-BSA concentration. In conclusion, an increase in AGE levels in vitro stimulates the secretion of EMMPRIN/MMPs, promotes the degradation of COL-I and reduces bone strength.

Fascin 1 promoted the growth and migration of non-small cell lung cancer cells by activating YAP/TEAD signaling.

Fascin 1 (Fascin actin-bundling protein 1) is an actin-binding protein. Although several studies have reported the dysregulation of Fascin 1 in non-small cell lung cancer (NSCLC), its functions in the progression of NSCLC and the related molecular mechanism were not fully understood. In this study, the expression of Fascin 1 in NSCLC tissues was determined using quantitative PCR (qPCR), and the roles of Fascin 1 in the progression of NSCLC were investigated. It was found that both the messenger RNA (mRNA) level and the protein level of Fascin 1 were upregulated in NSCLC tissues. Forced expression of Fascin 1 promoted the growth and migration of NSCLC cells, while knocking down the expression of Fascin 1 inhibited the growth, migration, and tumorigenesis of NSCLC cells. Mechanism studies showed that Fascin 1 increased the transcriptional activity of the YAP/TEAD (Yes-associated protein/TEA domain transcriptional factor) complex, and knocking down the expression of Fascin 1 attenuated the expression of target genes downstream the YAP/TEAD complex. In addition, MST1 interacted with Fascin 1. Taken together, Fascin 1 plays an oncogenic role in NSCLC by activating the transcriptional activity of the YAP/TEAD complex.

HDAC6 Regulates the Chaperone-Mediated Autophagy to Prevent Oxidative Damage in Injured Neurons after Experimental Spinal Cord Injury.

Hypoxia-ischemia- (HI-) induced oxidative stress plays a role in secondary pathocellular processes of acute spinal cord injury (SCI) due to HI from many kinds of mechanical trauma. Increasing evidence suggests that the histone deacetylase-6 (HDAC6) plays an important role in cell homeostasis in both physiological and abnormal, stressful, pathological conditions. This paper found that inhibition of HDAC6 accelerated reactive oxygen species (ROS) generation and cell apoptosis in response to the HI. Deficiency of HDAC6 hindered the chaperone-mediated autophagy (CMA) activity to resistance of HI-induced oxidative stress. Furthermore, this study provided the experimental evidence for the potential role of HDAC6 in the regulation of CMA by affecting HSP90 acetylation. Therefore, HDAC6 plays an important role in the function of CMA pathway under the HI stress induced by SCI and it may be a potential therapeutic target in acute SCI model.

Mesenchymal Stem Cell-Derived Exosomes Improve the Microenvironment of Infarcted Myocardium Contributing to Angiogenesis and Anti-Inflammation.

BACKGROUND/AIMS: Bone marrow mesenchymal stem cells (MSCs) widely applied for treating myocardial infarction face survival challenges in the inflammatory and ischemia microenvironment of acute myocardial infarction. The study hypothesized that MSC-derived exosomes play a significant role in improving microenvironment after acute myocardial infarction and aimed to investigate the paracrine effects of exosomes on angiogenesis and anti-inflammatory activity. METHODS: MSCs were cultured in DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin for 48 h. MSC-derived exosomes were isolated using ExoQuick-TC. Tube formation and T-cell proliferation assays were performed to assess the angiogenic potency of MSC-derived exosomes. Acute myocardial infarction was induced in Sprague-Dawley rats, and myocardium bordering the infarcted zone was injected at four different sites with phosphate-buffered saline (PBS, control), MSC-derived exosomes, and exosome-depleted MSC culture medium. RESULTS: MSC-derived exosomes significantly enhanced the tube formation of human umbilical vein endothelial cells, impaired T-cell function by inhibiting cell proliferation in vitro, reduced infarct size, and preserved cardiac systolic and diastolic performance compared with PBS markedly enhancing the density of new functional capillary and hence blood flow recovery in rat myocardial infarction model. CONCLUSIONS: Exosomes stimulate neovascularization and restrain the inflammation response, thus improving heart function after ischemic injury.

Follistatin-like 1 attenuates differentiation and survival of erythroid cells through Smad2/3 signaling.

Hematopoiesis is a complex process tightly controlled by sets of transcription factors in a context-dependent and stage-specific manner. Smad2/3 transcription factor plays a central role in differentiation and survival of erythroid cells. Here we report that follistatin-like 1 (FSTL1) treatment impairs hemin-induced erythroid differentiation and cell survival. FSTL1 differentially regulates transforming growth factor beta (TGF-ß) and bone morphogenetic protein (BMP) signaling. Blockade of Smad2/3 signaling with the ALK5/type I TGF-ßR kinase inhibitor, SB-525334, was efficacious for rescue of erythroid differentiation blockage and apoptosis. Reversely, activation of Smad1/5/8 signaling with BMP4 cannot rescue FSTL1-mediated erythroid differentiation blockage and apoptosis. Collectively, these data provide mechanistic insight into the regulation of erythropoiesis by FSTL1 signaling and lay a foundation for exploring FSTL1 signaling as a therapeutic target for anemia.

Determination of co-administrated opioids and benzodiazepines in urine using column-switching solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Co-administration of opioids with benzodiazepines is very common around the world. A semi-automated method was developed for the determination of four opioids and two benzodiazepines as well as their metabolites (including glucuronide metabolites) in human urine, based on on-line column-switching-solid-phase extraction (CS-SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CS-SPE was performed by loading 200µL of urine sample to an Oasis HLB cartridge. Detection was achieved using a LC-MS/MS system equipped with an electrospray ionization source (ESI). For unequivocal identification and confirmation, two selected reaction monitoring transitions were registered for each compound, and no co-elution of interferences was observed at the expected retention time. Significant ion suppressions were observed for most analytes during chromatographic runs, but isotope-labeled internal standards (ISs) were used and found to be useful to compensate for the determination error caused by the matrix effect. The assay's linearity ranged from 1-20ng/mL to 800-1000ng/mL for 23 compounds, except for lorazepam (LOR), whose linearity was in the range of 1-100ng/mL. This method showed to be precise and accurate. The relative standard deviation (RSD) % values of within-run precision, between-run precision and total precision were not greater than 10.4% (n=3), 12.9% (n=5) and 15.1% (n=15), respectively. Accuracy values were in the range of 87.5-110%. Limits of detection (LODs) ranged from 0.2ng/mL to 5ng/mL, and limits of quantification (LOQs) ranged from 1ng/mL to 20ng/mL. The method was applied to the assay of 12 samples from forensic cases, which exemplified the co-administration of benzodiazepines (BZDs) by some heroin abusers. This method was of high sensitivity, selectivity and reliability, minimum sample manipulation, semi-automation, and fairly high throughput (analysis time per sample was 20min). The method developed will be useful for the detection of co-administrated drugs and the study of the interactions of BDZs with opioids.

Screening of drugs of abuse and toxic compounds in human whole blood using online solid-phase extraction and high-performance liquid chromatography with time-of-flight mass spectrometry.

A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid-phase extraction with liquid chromatography coupled to time-of-flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid-phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time-of-flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full-scan (m/z 50-800) mode using an MS(E) acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5-45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.

Injectable biodegradable hybrid hydrogels based on thiolated collagen and oligo(acryloyl carbonate)-poly(ethylene glycol)-oligo(acryloyl carbonate) copolymer for functional cardiac regeneration.

Injectable biodegradable hybrid hydrogels were designed and developed based on thiolated collagen (Col-SH) and multiple acrylate containing oligo(acryloyl carbonate)-b-poly(ethylene glycol)-b-oligo(acryloyl carbonate) (OAC-PEG-OAC) copolymers for functional cardiac regeneration. Hydrogels were readily formed under physiological conditions (37°C and pH 7.4) from Col-SH and OAC-PEG-OAC via a Michael-type addition reaction, with gelation times ranging from 0.4 to 8.1 min and storage moduli from 11.4 to 55.6 kPa, depending on the polymer concentrations, solution pH and degrees of substitution of Col-SH. The collagen component in the hybrid hydrogels retained its enzymatic degradability against collagenase, and the degradation time of the hydrogels increased with increasing polymer concentration. In vitro studies showed that bone marrow mesenchymal stem cells (BMSCs) exhibited rapid cell spreading and extensive cellular network formation on these hybrid hydrogels. In a rat infarction model, the infarcted left ventricle was injected with PBS, hybrid hydrogels, BMSCs or BMSC-encapsulating hybrid hydrogels. Echocardiography demonstrated that the hybrid hydrogels and BMSC-encapsulating hydrogels could increase the ejection fraction at 28 days compared to the PBS control group, resulting in improved cardiac function. Histology revealed that the injected hybrid hydrogels significantly reduced the infarct size and increased the wall thickness, and these were further improved with the BMSC-encapsulating hybrid hydrogel treatment, probably related to the enhanced engraftment and persistence of the BMSCs when delivered within the hybrid hydrogel. Thus, these injectable hybrid hydrogels combining intrinsic bioactivity of collagen, controlled mechanical properties and enhanced stability provide a versatile platform for functional cardiac regeneration.

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction.

It is still unclear whether the timing of intracoronary stem cell therapy affects the therapeutic response in patients with myocardial infarction. The natural course of healing the infarction and the presence of putative homing signals within the damaged myocardium appear to favor cell engraftment during the transendothelial passage in the early days after reperfusion. However, the adverse inflammatory environment, with its high oxidative stress, might be deleterious if cells are administered too early after reperfusion. Here we highlight several aspects of the timing of intracoronary stem cell therapy. Our results showed that transplantation of bone marrow mesenchymal stem cells at 2-4 weeks after myocardial infarction is more favorable for reduction of the scar area, inhibition of left ventricular remodeling, and recovery of heart function. Coronary injection of autologous bone marrow mesenchymal stem cells at 2-4 weeks after acute myocardial infarction is safe and does not increase the incidence of complications.