Discovery of novel serum biomarkers for diagnosing and predicting postmenopausal osteoporosis patients by 4D-label free protein omics.

We aimed to identify protein biomarkers that could rapidly and accurately diagnose osteoporosis patients (OPs) using a highly sensitive proteomic immunoassay. Four-dimensional (4D) label-free proteomics analysis was performed to determine the differentially expressed proteins in serum collected from 10 postmenopausal osteoporosis patients and 6 non-osteoporosis patients. The ELISA method was used to select the predicted proteins for verification. Serum was taken from 36 postmenopausal osteoporosis patients and 36 healthy individuals from normal postmenopausal women. Receiver operating characteristic (ROC) curves were used to determine the diagnostic potential of this method. We validated the expression of these six proteins using ELISA. The CDH1, IGFBP2, and VWF of osteoporosis patients were significantly higher than those of the normal group. PNP was significantly lower than that in the normal group. And using ROC curve calculation, serum CDH1 had a cut-off of 3.78 ng/mL with a sensitivity of 84.4%, and PNP had a cut-off of 944.32 ng/mL with 88.9% sensitivity. These outcomes suggest that serum-level CHD1 and PNP have the potential power as effective indicators for the diagnosis of PMOP. Our results suggest that CHD1 and PNP might be associated with the pathogenesis of OP and would be helpful in diagnosing OP. Therefore, CHD1 and PNP may act as potential key markers in OP.

The interaction protein of SORBS2 in myocardial tissue to find out the pathogenic mechanism of LVNC disease.

BACKGROUND: Left ventricular noncompaction cardiomyopathy (LVNC) is a cardiac disorder characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. Sorbin and SH3 domain-containing protein 2 (SORBS2) converges on the actin and microtubule cytoskeleton. Here, we investigated the proteins interacting with SORBS2 to elucidate the pathogenic mechanism of LVNC. As reported in previous studies, SORBS2 enhances the occurrence of LVNC by potentiating heart failure, but the specific mechanism remains unclear. METHODS: Building from our previous finding of elevated SORBS2 levels in LVNC hearts, we screened for proteins interacting with SORBS2 by proteomics and conducting IP experiments. Co-IP and immunofluorescence were used to verify the effects. RESULTS: We selected several proteins with high scores and high coverage that could be closely related to SORBS2 according to earlier reports showing a correlation with LVNC for verification. We finally obtained several proteins that were related to the pathogenesis of LVNC and also interacted with SORBS2, such as α-actinin, ß-tubulin, MYH7, FLNA, MYBPC3, YWHAQ and DES, and YWHAQ was the most associated. CONCLUSIONS: We focused on the YWHAQ protein, and we identified a novel mechanism through which SORBS2 interacts with YWHAQ, having a negative effect on the cell cycle, potentially leading to LVNC.

Suppression of Myocardial Hypoxia-Inducible Factor-1α Compromises Metabolic Adaptation and Impairs Cardiac Function in Patients With Cyanotic Congenital Heart Disease During Puberty.

BACKGROUND: Cyanotic congenital heart disease (CCHD) is a complex pathophysiological condition involving systemic chronic hypoxia (CH). Some patients with CCHD are unoperated for various reasons and remain chronically hypoxic throughout their lives, which heightens the risk of heart failure as they age. Hypoxia activates cellular metabolic adaptation to balance energy demands by accumulating hypoxia-inducible factor 1-α (HIF-1α). This study aims to determine the effect of CH on cardiac metabolism and function in patients with CCHD and its association with age. The role of HIF-1α in this process was investigated, and potential therapeutic targets were explored. METHODS: Patients with CCHD (n=25) were evaluated for cardiac metabolism and function with positron emission tomography/computed tomography and magnetic resonance imaging. Heart tissue samples were subjected to metabolomic and protein analyses. CH rodent models were generated to enable continuous observation of changes in cardiac metabolism and function. The role of HIF-1α in cardiac metabolic adaptation to CH was investigated with genetically modified animals and isotope-labeled metabolomic pathway tracing studies. RESULTS: Prepubertal patients with CCHD had glucose-dominant cardiac metabolism and normal cardiac function. In comparison, among patients who had entered puberty, the levels of myocardial glucose uptake and glycolytic intermediates were significantly decreased, but fatty acids were significantly increased, along with decreased left ventricular ejection fraction. These clinical phenotypes were replicated in CH rodent models. In patients with CCHD and animals exposed to CH, myocardial HIF-1α was upregulated before puberty but was significantly downregulated during puberty. In cardiomyocyte-specific Hif-1α-knockout mice, CH failed to initiate the switch of myocardial substrates from fatty acids to glucose, thereby inhibiting ATP production and impairing cardiac function. Increased insulin resistance during puberty suppressed myocardial HIF-1α and was responsible for cardiac metabolic maladaptation in animals exposed to CH. Pioglitazone significantly reduced myocardial insulin resistance, restored glucose metabolism, and improved cardiac function in pubertal CH animals. CONCLUSIONS: In patients with CCHD, maladaptation of cardiac metabolism occurred during puberty, along with impaired cardiac function. HIF-1α was identified as the key regulator of cardiac metabolic adaptation in animals exposed to CH, and pubertal insulin resistance could suppress its expression. Pioglitazone administration during puberty might help improve cardiac function in patients with CCHD.

Cold-inducible RNA binding protein agonist enhances the cardioprotective effect of UW solution during extended heart preservation.

In heart transplantation, time restriction is an unavoidable thorny problem during cardiac transport. Cold storage is an important organ preservation method in donor heart transport. Cold-inducible RNA binding protein (CIRBP) has been proven to play a protective role under cold stress. In this study, we investigated the role of CIRBP in hypothermic cardioprotection during heart preservation in UW solution and explored a new approach to extend the heart preservation time. Cirbp-knockout (Cirbp-/- ), Cirbp-transgenic (Cirbp-Tg), and wild-type rats were, respectively, randomized into two groups based on various heart preservation times (6 or 12-hour group) (n = 8 per group). After preservation in UW solution, all hearts were mounted on a Langendorff apparatus and underwent measurement of cardiac parameters, histological analysis, and molecular study. Within the 6-hour preservation group, no significant difference was found in cardiac functions and histological changes between different rat species. However, after 12 hours of preservation, Cirbp-/- rat hearts showed more apoptosis and worse cardiac function, but less apoptosis and better cardiac function were observed in Cirbp-Tg rat hearts. Furthermore, we found CIRBP-mediated cardiac ubiquinone (CoQ10 ) biosynthesis plays an important role in extending heart preservation, and ubiquinone biosynthesis protein COQ9 was an essential down-stream regulator during this process. Finally, we found that zr17-2, a CIRBP agonist, could enhance the expression of CIRBP, which further enhances the synthesis of CoQ10 and promotes scavenging of reactive oxygen species and ATP production to extend heart preservation. This study demonstrated that CIRBP-enhanced CoQ10 biosynthesis during hypothermic heart preservation and zr17-2-supplemented UW solution could be a promising approach to ameliorate heart damage and extend heart preservation during cardiac transport.

Elevated myocardial SORBS2 and the underlying implications in left ventricular noncompaction cardiomyopathy.

BACKGROUND: Left ventricular noncompaction cardiomyopathy (LVNC) is a hereditary heart disease characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. The guidelines for management of LVNC patients aim to improve quality of life by preventing cardiac heart failure. However, the mechanism underlying LVNC-associated heart failure remains poorly understood. METHODS: Using protein mass spectrometry analysis, we established that Sorbin And SH3 Domain Containing 2 (SORBS2) is up-regulated in LVNC hearts without changes to structure proteins. We conducted in vivo experiments wherein the heart tissues of wild-type mice were injected with an AAV9 vector to overexpress SORBS2, followed by analysis using echocardiography, T-tubule analysis and Ca2+ imaging to identify functional and morphological changes. In addition, we analyzed the function and structure of SORBS2 overexpressing human embryonic stem cell (hESC) derived cardiomyocytes (hESC-CM) via immunoblotting, immunohistochemistry, immunofluorescence, and confocal Ca2+ imaging. FINDINGS: LVNC myocardial tissues feature strongly elevated expression of SORBS2, microtubule densification and redistribution of Junctophilin 2 (JP2). SORBS2 interacts with ß-tubulin, promoting its polymerization in 293T cells and hESC-derived CMs. In vivo, cardiac dysfunction, ß-tubulin densification, JP2 translocation, T-tubule disorganization and Ca2+ handling dysfunction were observed in mice overexpressing SORBS2. INTERPRETATION: We identified a novel mechanism through which SORBS2 interacts with ß-tubulin and promotes microtubule densification, eventually effecting JP2 distribution and T-tubule, potentially contributing to heart failure in LVNC disease. FUND: This work was supported by a CAMS Initiative for Innovative Medicine grant (CAMS-I2M, 2016-I2M-1-015 to Y.J.Wei).

Transthoracic Pulmonary Artery Denervation for Pulmonary Arterial Hypertension.

Objective- Pulmonary arterial hypertension is characterized by progressive pulmonary vascular remodeling and persistently elevated mean pulmonary artery pressures and pulmonary vascular resistance. We aimed to investigate whether transthoracic pulmonary artery denervation (TPADN) attenuated pulmonary artery (PA) remodeling, improved right ventricular (RV) function, and affected underlying mechanisms. We also explored the distributions of sympathetic nerves (SNs) around human PAs for clinical translation. Approach and Results- We identified numerous SNs in adipose and connective tissues around the main PA trunks and bifurcations in male Sprague Dawley rats, which were verified in samples from human heart transplant patients. Pulmonary arterial hypertensive rats were randomized into TPADN and sham groups. In the TPADN group, SNs around the PA trunk and bifurcation were completely and accurately removed under direct visualization. The sham group underwent thoracotomy. Hemodynamics, RV function, and pathological changes in PA and RV tissues were measured via right heart catheterization, cardiac magnetic resonance imaging, and pathological staining, respectively. Compared with the sham group, the TPADN group had lower mean pulmonary arterial pressures, less PA and RV remodeling, and improved RV function. Furthermore, TPADN inhibited neurohormonal overactivation of the sympathetic nervous system and renin-angiotensin-aldosterone system and regulated abnormal expressions and signaling of neurohormone receptors in local tissues. Conclusions- There are numerous SNs around the rat and human main PA trunks and bifurcations. TPADN completely and accurately removed the main SNs around PAs and attenuated pulmonary arterial hypertensive progression by inhibiting excessive activation of the sympathetic nervous system and renin-angiotensin-aldosterone system neurohormone-receptor axes.

Fluoxetine attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage: a possible role for the regulation of TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND: Neuroinflammation is closely associated with functional outcome in subarachnoid hemorrhage (SAH) patients. Our recent study demonstrated that fluoxetine inhibited NLRP3 inflammasome activation and attenuated necrotic cell death in early brain injury after SAH, while the effects and potential mechanisms of fluoxetine on neuroinflammation after SAH have not been well-studied yet. METHODS: One hundred and fifty-three male SD rats were subjected to the endovascular perforation model of SAH. Fluoxetine (10 mg/kg) was administered intravenously at 6 h after SAH induction. TAK-242 (1.5 mg/kg), an exogenous TLR4 antagonist, was injected intraperitoneally 1 h after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, immunofluorescence/TUNEL staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were performed. RESULTS: Fluoxetine administration attenuated BBB disruption, brain edema, and improved neurological function after SAH. In addition, fluoxetine alleviated the number of Iba-1-positive microglia/macrophages, neutrophil infiltration, and cell death. Moreover, fluoxetine reduced the levels of pro-inflammatory cytokines, downregulated the expression of TLR4 and MyD88, and promoted the nuclear translocation of NF-κB p65, which were also found in rats with TAK-242 administration. Combined administration of fluoxetine and TAK-242 did not enhance the neuroprotective effects of fluoxetine. CONCLUSION: Fluoxetine attenuated neuroinflammation and improved neurological function in SAH rats. The potential mechanisms involved, at least in part, TLR4/MyD88/NF-κB signaling pathway.

Inhibiting of RIPK3 attenuates early brain injury following subarachnoid hemorrhage: Possibly through alleviating necroptosis.

Necroptosis is an inflammatory form of cell death that depends on receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) and displays the morphological characteristics of necrosis. To date, it is unclear to what extent necroptosis contributes to subarachnoid hemorrhage (SAH) induced brain injury. The present study aimed to investigate the RIPK3-mediated necroptosis and the effects of the RIPK3 selective inhibitor GSK'872 in early brain injury following SAH. After SAH, RIPK3 expression increased as early as 6 h and peaked at 72 h. Double immunofluorescence staining revealed that RIPK3 was mainly located in neurons. Most necrotic cells were neurons, which were further confirmed by TEM. Intracerebroventricular injection of GSK'872 (25 mM) could attenuate brain edema and improve neurological function following SAH and reduce the number of necrotic cells. In addition, GSK'872 could also decrease the protein levels of RIPK3 and MLKL, and cytoplasmic translocation and expression of HMGB1, an important pro-inflammatory protein. Taken together, the current study provides the new evidence that RIPK3-mediated necroptosis is involved in early brain injury and GSK'872 decreases the RIPK3-mediated necroptosis and subsequent cytoplasmic translocation and expression of HMGB1, as well as ameliorates brain edema and neurological deficits.