Growth Factor Stimulation Research Articles

Serum Insufficiency Induces RANKL-Independent Osteoclast Formation during Developing Ischemic ONFH.

Blood supply interruption induces hypoxia and reduces serum provision to cause ischemia-induced osteonecrosis, including avascular osteonecrosis of the femoral head (ONFH). Oxygen deficiency (hypoxia) is known to induce different expression patterns in osteoblasts and osteoclasts, which have been extensively studied. However, the effects of serum insufficiency in nutrients, growth factors, and hormones on osteoblast and osteoclast activity in the damaged area and nearby regions remain poorly understood. In this study, the expression of osteoblast and osteoclast marker proteins was elucidated through in vitro and ex vivo studies. The results indicate that serum insufficiency accelerates the formation of monocyte-derived osteoclasts. The combined effect of serum insufficiency and hypoxia (mimicking ischemia) suppressed the activity of alkaline phosphatase and calcification in osteoblasts after the stimulation of osteogenic growth factors. Serum insufficiency increased the activity of tartrate-resistant acid phosphatase, expression of phosphorylated extracellular signal-regulated kinases, and production of reactive oxygen species in monocyte-derived osteoclasts in the absence of receptor activator of nuclear factor kappa-Β ligand stimulation. The findings indicate that changes in the expression of osteoblast and osteoclast markers in necrotic bone extracts were similar to those observed during an in vitro study. These results also suggest that serum insufficiency may be involved in the regulation of osteoclast formation in patients with ONFH.

A Bilayered, Electrospun Poly(Glycerol-Sebacate)/Polyurethane-Polyurethane Scaffold for Engineering of Endothelial Basement Membrane.

In the cardiovascular system, heart valves and vessels are subjected to continuous cyclic mechanical loadings due to the pulsatile nature of blood flow. Hence, in leveraging tissue engineering (TE) strategies to regenerate such a system, the candidate scaffold should not only be biocompatible with the desired biodegradation rate, but it should also be mechanically competent to provide a supportive structure for facilitating stem cells retention, growth, and differentiation. To this end, herein, we introduced a novel scaffold composed of poly(glycerol-sebacate) (PGS) and polyurethane (PU), which comprises of two layers: an electrospun pure PU layer beneath another electrospun PGS/PU layer with a different ratio of PGS to PU (3:2, 1:1, 2:3 Wt:Wt). The electrospun PGS/PU-PU scaffold was mechanically competent and showed intended hydrophilicity and a good biodegradation rate. Moreover, the PGS/PU-PU scaffold indicated cell viability and proliferation within ten days of in vitro cell culture and upon 7 day vascular endothelial growth factor (VEGF) stimulation, supported endothelial differentiation of mesenchymal stem cells by significant overexpression of platelet-endothelial cell adhesion molecule-1, von Willebrand factor, and VEGF receptor 2. The results of this study could be implemented in cardiovascular TE strategies when regeneration of blood vessel or heart valve is desired.

Chemopreventive effects and anti-tumorigenic mechanisms of 2,6-dimethoxy-1,4-benzoquinone, a constituent of Vitis coignetiae Pulliat (crimson glory vine, known as yamabudo in Japan), toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice

Previously, we isolated and identified anti-mutagenic and anti-inflammatory components from Vitis coignetiae (crimson glory vine, known as yamabudo in Japan) as 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid. We also reported that the oral intake of a partially purified fraction from yamabudo juice (yamabudo-fr) or DBQ affords significant protection against two-stage skin carcinogenesis in mice. In this study, we found that oral intake of yamabudo-fr or DBQ affords significant protection against a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse model of lung tumorigenesis. Furthermore, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ. NNK is known to be a DNA-methylating and alkylating agent; thus, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ in relation to DNA methylation. Pretreatment with yamabudo-fr or DBQ dose-dependently decreased formation of O6-methylguanine and N7-methylguanine in DNA of the A549 human lung epithelial-like cell line treated with a methylating agent, 1-methyl-3-nitro-1-nitrosoguanidine. Yamabudo juice and DBQ inhibited the mutagenicity of NNK in the Ames test using Salmonella typhimurium TA1535 but not S. typhimurium YG7108, an alkylguanine DNA alkyltransferase-deficient strain (same as TA1535 but Δadast::Kmr, Δogtst::Cmr). Yamabudo juice and DBQ might accelerate the repair of DNA damage caused by NNK and reduce DNA damage to cells. We also investigated the effects of yamabudo juice and DBQ on signaling pathways in A549 cells. With or without epidermal growth factor stimulation, phosphorylation of Erk1/2, Akt and Stat3 in A549 cells was significantly decreased in the presence of yamabudo juice or DBQ, indicating that yamabudo juice and DBQ suppressed PI3K/AKT, MAPK/ERK and JAK/STAT3 signaling pathways. These results suggest that both initiation and growth/progression steps in carcinogenesis, especially anti-oxidant effects, stimulation of repair of alkyl DNA adducts and suppressed growth signaling pathways are potential anti-tumorigenic targets of yamabudo juice and DBQ in NNK-induced lung tumorigenesis.

Role of stimulating growth factor 2 and galectin-3 in predicting the ventricular tachyarrhythmias in patients with ischemic cardiomyopathy

Aim. To assess the role of stimulating growth factor 2 (ST-2) and galectin-3 in predicting the ventricular tachyarrhythmias (VTA) in patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35%.Material and methods. The study included 40 patients (men, 36 (90,0%); median age, 64,5 [57,5; 68,5] years) with CAD, NYHA class IIIII heart failure, LVEF <35%, indications for implantable cardioverter defibrillator (ICD) (primary prevention of sudden cardiac death). Prior to ICD implantation, patients were measured for serum ST-2 and galectin-3 levels. For 18 months, patients were assessed for arrhythmic events recorded in the ICD memory.Results. The 1st group consisted of 10 (25,0%) patients who had VTA episodes, arrested by antitachycardia pacing or shock during 18-month follow-up. The 2nd group consisted of 30 (75,0%) patients without VTA episodes. It was found that ST-2 concentration >22,48 ng/ml (p=0,02) and galectin-3 >10,95 ng/ml (p=0,009) corresponded to appropriate ICD discharge. Multivariate ROC analysis demonstrated that the only independent predictor of VTA was ST-2 elevation (odds ratio, 1,11; 95% CI, 1,01-1,21; p=0,023).Conclusion. An increase in the concentration of both ST-2 >22,48 ng/ml and galectin-3 >10,95 ng/ml had a high predictive value in assessing the VTA risk in patients with ischemic cardiomyopathy. In multivariate analysis, an increase in ST-2 >22,48 ng/ml was an independent predictor of VTA.

Mesenchymal stem cell-based bioengineered constructs enhance vaginal repair in ovariectomized rhesus monkeys

Transvaginal meshes repair for treating pelvic organ prolapse (POP) was halted by the U. S. Food and Drug Administration (FDA) because they can lead to severe complications. Therefore, investigations of new therapeutic strategies are urgently needed. Cell-based regenerative therapy holds great promise for the repair and restoration of damaged tissue. Here, we generated a bioengineered graft by seeding human umbilical cord mesenchymal stem cells (HUMSCs) on bioscaffolds to reconstruct the damaged vagina. In the in vitro study, HUMSCs proliferated well and the density was appropriate after 5 days of culture. Besides, we demonstrated that the differentiation potential of HUMSCs was maintained with external growth factor stimulation. The complete transcriptomic profile of HUMSCs revealed that HUMSCs cultured on grafts produced significantly higher levels of proangiogenic cytokines than cells cultured in tissue culture plates (TCPs). Three months after implantation of the bioengineered grafts into ovariectomized (OVX) rhesus monkeys via sacrocolpopexy, extracellular matrix reorganization, large muscle bundle formation, angiogenesis and, mechanical properties of the vagina were enhanced. To our knowledge, this is the first demonstration of the utility of stem cell-based bioengineered grafts for repairing damaged vaginal tissue in rhesus monkeys. These results elucidate a new approach for vagina repair and provide new ideas for treating POP.

Isoform‐Selective Regulation of Akt1 and Akt2 by Interdomain Interaction

The serine/threonine protein kinase PKB/Akt is a key signal transducer in a wide range of cellular responses and a prominent therapeutic target. The three highly homologous isoforms of Akt regulate critical and distinct physiological functions in cell growth, proliferation, and metabolism. However, the mechanisms that determine isoform-specific Akt signaling remain largely unclear. Here we examined the mechanisms by which Akt1 and Akt2, the most broadly expressed isoforms, are differentially activated upon growth factor stimulation. Using a newly developed FRET-based reporter of Akt2 activation (ReAktion2) along with a previously developed reporter of Akt1 activation (ReAktion), we found that growth factor triggers a conformational change in both Akt isoforms at the plasma membrane, which is critically dependent on activation-loop phosphorylation. Interestingly, the allosteric pan-Akt inhibitor, MK-2206, appears to selectively induce a conformational change in Akt2, but not Akt1 at the plasma membrane. This prompts us to further examine whether inter-domain interactions differ between Akt1 and Akt2. Our preliminary results suggest Akt1 activation enhances the interaction between its PH and linker-catalytic domains, which is dependent on phosphorylation of T308 within the catalytic domain, whereas this induced interdomain interaction is absent in Akt2. Furthermore, the different interdomain interaction of Akt isoforms may lead to different membrane departure, as single cell analysis of plasma membrane translocation revealed that Akt2 accumulates at the plasma membrane to a greater extent than Akt1. Taken together, our results suggest that distinct modes of interactions between the PH and linker-catalytic domains modulate the isoform-specific activation of Akt.

Respective, Time‐dependent Phosphorylation Modules Shaping Phosphoproteome Abundance and Turnover

Protein phosphorylation mediates protein-protein interactions and alters protein structure, stability, and subcellular localization—frequently in a modification site-specific manner. However, how phosphorylation impacts protein lifetime remains to be illustrated. We have devised a proteomic method, DeltaSILAC, to quantitatively assess the influence of site-specific phosphorylation on the turnover of thousands of proteins in live cells (1). We discovered many phosphosites prolonging protein lifetime, challenging the previous notion that phosphorylation mostly accelerates protein degradation. Extending this research analyzing steady-state cells, we herein aimed to characterize how the timing and duration of phosphorylation signaling dedicate cell phenotypes. In a classic signaling system, the differential stimulation of epidermal and nerve growth factors (EGF and NGF) can determine the cell fate. Whereas EGF “transiently” stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation, NGF stimulation induces “sustained” activation of MAPKs and cell differentiation. Herein, we applied both DeltaSILAC and label-free phosphoproteomics. We developed DeltaSILAC with a pulse labeling using stable isotope-labeled amino acids in cells (pSILAC) accompanying the EGF and NGF stimulations on PC12 cells. Our time-course design embraced both short- and long-term points: 0-sec, 15-sec, 30-sec, 1-min, 2-min, 5-min, 10-min, 30-min, 1-hour, 2 hour, 4-hour, 8-hour, 12-hour, 24-hour, 48-hour, and 72-hour. We also generated matching protein- and mRNA- profiles after 30 mins following stimulations, so that phosphorylation regulations can be dissected from gene expression dynamics. We utilized a novel peptide-level matching algorithm so that both abundance and lifetime can be measured and resolved for each phosphosite. By using a reproducible mass spectrometry method named data-independent acquisition (DIA-MS), collectively, we quantified 40,000 phosphopeptides and 9,000 proteins throughout the experiment, both globally recapitulated the “transient” and “sustained” patterns of EGF and NGF signaling. We discovered specific tyrosine phosphorylation as early as 15-30 seconds. Interesting EGFR phosphosites activated by NGF treatment were ascribed to P38 kinase activity. The modeling of our unique time-series datasets of long-term time points indicates that phosphosites involved in EGF/NGF signaling transduction and mRNA processing shortened the corresponding protein lifetime, whereas phosphosites in the apoptotic process stabilized protein expression. We conclude that while early phosphorylation events initiate signaling functions through phosphate-dependent transfer, the longer-lasting phosphorylation events could shape proteome stability and proteostasis in cell fate determination.1. C. Wu et al., Global and Site-Specific Effect of Phosphorylation on Protein Turnover . Developmental cell 10.1016/j.devcel.2020.10.025 (2020).

Sensitive Akt biosensor and expansion microscopy reveal regulation of Akt at the lysosome

The phosphoinositide 3-kinase (PI3K)/Akt pathway regulates a wide range of cellular processes, including cell survival, growth and metabolism. As a critical component of this pathway, the serine/threonine protein kinase Akt activates the downstream component mechanistic target of rapamycin complex 1 (mTORC1) through spatial control of the TSC complex at the lysosome but also has downstream targets throughout the cell. However, the specific spatial organization of Akt activity and the underlying regulatory mechanisms, including the mechanism controlling its activity at the lysosome, are not clearly understood. To interrogate the regulation of Akt at subcellular locations, we developed a new genetically encoded fluorescent Akt activity biosensor. Coupling an Akt-specific substrate peptide and a phosphoamino acid binding domain to either side of circularly permutated GFP via optimized linkers yielded an excitation-ratiometric Akt activity reporter (ExRai-AktAR2) that exhibits excitation maxima at both ~400 nm and ~500 nm, along with a single emission peak near 515 nm, and undergoes an excitation ratio change of about 2 fold. Genetically targeted ExRai-AktAR2 revealed widespread Akt activity at different subcellular locations, including cytoplasm, plasma membrane, golgi, and nucleus. Using a lysosomally targeted ExRai-AktAR2, we showed that growth factor stimulation induces Akt activity at the lysosomal surface, which is dependent on presence of 3-phosphoinositides (3-PIs) on the lysosome membrane. Further dissection using pharmacological perturbation, genetically targeted manipulation of 3-PIs and expansion microscopy showed that lysosomal 3-PIs, which are dependent on dynamin-mediated endocytosis, are critical for recruiting Akt to the lysosome, resulting in accumulation of Akt activity and mTORC1 activity on the lysosomal surface. Thus, 3-PIs, a class of critical lipid second messengers that are mainly found in the plasma membrane, accumulate on lysosomal membrane to direct a multi-faceted kinase to organize lysosome-specific signaling for local activation of downstream effectors, showcasing intricate spatial organization and regulation of a key signaling pathway.

Targeting STAT3: A crucial modulator of sepsis.

Signal transducer and activator of transcription 3 (STAT3) is a cellular signal transcription factor that has recently attracted a great deal of attention. It can trigger a variety of genes transcription in response to cytokines and growth factors stimulation, which plays an important role in many cellular biological processes involved in anti/proinflammatory responses. Sepsis is a life-threatening organ dysfunction resulting from dysregulated host responses to infection. As a converging point of multiple inflammatory responses pathways, accumulating studies have presented the elaborate network of STAT3 in sepsis pathophysiology; these results generally indicate a promising therapeutic application for targeting STAT3 in the treatment of sepsis. In the present review, we evaluated the published literature describing the use of STAT3 in the treatment of experimental and clinical sepsis. The information presented here may be useful for the design of future studies and may highlight the potential of STAT3 as a future biomarker and therapeutic target for sepsis.

Carbohydrate metabolism disorders in patients with heart failure: data from the local registry

Aim. To study the prevalence of carbohydrate metabolism disorders in patients with heart failure (HF) hospitalized in the city HF center.Material and methods. According to the local registry, the study sequentially included 183 patients (99 men and 84 women) hospitalized in the Nizhny Novgorod city HF center from September 1, 2019. The examination and treatment were carried out in accordance with the current clinical guidelines. In the first 48 hours after hospitalization, the concentration of the N-terminal pro-brain natriuretic peptide, soluble stimulating growth factor 2 (sST2), neutrophil gelatinase-associated lipocalin, cystatin C, blood creatinine was determined. The glomerular filtration rate was calculated using the СKDEPI equation. To assess the carbohydrate metabolism disorders, all patients were studied for fasting plasma glucose, glycated hemoglobin (HbA1c) and fructosamine. Statistical data processing was carried out using the R statistics package (R Core Team (2019)).Results. The incidence of carbohydrate metabolism disorders among patients withdecompensated HF was 75,89%, including previously diagnosed type 2 diabetes in 31,25%, newly diagnosed dysglycemia in 44,64% of patients. Less than one fourth of patients had normal parameters of carbohydrate metabolism according to HbA1c, fructosamine and fasting plasma glucose. The severity of carbohydrate metabolism disorders was significantly correlated with the severity of HF according to the following criteria: 6-minute walk test, HF functional class, sST2 level, and some parameters of cardiac remodeling. Among the criteria used for carbohydrate metabolism disorders, the HbA1c level was most closely associated with the criteria for HF severity.Conclusion. Carbohydrate metabolism disorders in HF patients are widespread and underdiagnosed during routine examination. The interrelation of carbohydrate metabolism parameters and indicators of HF severity is rationale for active detection of dysglycemia in these patients in order to potentially influence the prognosis.

Rosuvastatin Inhibits the Apoptosis of Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells by Inhibiting p38 via Autophagy.

The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1β (IL-1β) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis.

Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.

In the lungs, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological responses remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lungs and thus assumed to be the same as the one acting on the alveolar epithelium. Existing in vitro models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced in vitro vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature in vitro. We studied the effects of the cyclic strain on a perfusable 3D vasculature, made of either human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The in vitro 3D vessels underwent both in vivo-like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with the 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however, we observed several phenomena in the 3D model that the 2D model was unable to predict. Angiogenic sprouting induced by VEGF decreased significantly in the presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast to our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer, which truly recapitulates the in vivo situation.

Grb2 binding induces phosphorylation-independent activation of Shp2

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.

Gene Expression Profiling and Biofunction Analysis of HepG2 Cells Targeted by Crocetin.

Crocetin is a carotenoid extracted from Gardenia jasminoides, one of the most popular traditional Chinese medicines, which has been used in the prevention and treatment of various diseases. The present study is aimed at clarifying the effect of crocetin on gene expression profiling of HepG2 cells by RNA-sequence assay and further investigating the molecular mechanism underlying the multiple biofunctions of crocetin based on bioinformatics analysis and molecular evidence. Among a total 23K differential genes identified, crocetin treatment upregulated the signals of 491 genes (2.14% of total gene probes) and downregulated the signals of 283 genes (1.24% of total gene probes) by ≥2-fold. The Gene Ontology analysis enriched these genes mainly on cell proliferation and apoptosis (BRD4 and DAXX); lipid formation (EHMT2); cell response to growth factor stimulation (CYP24A1 and GCNT2); and growth factor binding (ABCB1 and ABCG1), metabolism, and signal transduction processes. The KEGG pathway analysis revealed that crocetin has the potential to regulate transcriptional misregulation, ABC transporters, bile secretion, alcoholism, systemic lupus erythematosus (SLE), and other pathways, of which SLE was the most significantly disturbed pathway. The PPI network was constructed by using the STRING online protein interaction database and Cytoscape software, and 21 core proteins were obtained. RT-qPCR datasets serve as the solid evidence that verified the accuracy of transcriptome sequencing results with the same change trend. This study provides first-hand data for comprehensively understanding crocetin targeting on hepatic metabolism and its multiple biofunctions.

The Value of Biomarkers in the Diagnosis and Prognosis of Heart Failure in Older Age

The search for reliable algorithms for diagnosing heart failure with preserved left ventricular ejection fraction (LVEF) in elderly patients is an urgent problem due to the low specificity of clinical manifestations and the peculiarities of involutive processes occurring in the human body. As an alternative diagnostic approach, it is possible to determine in the blood laboratory biochemical markers — a promising method of diagnosis, prognosis and control of the effectiveness of treatment. The article examines the significance of myocardial stress markers (brain natriuretic peptide, N-terminal brain natriuretic peptide, median fragment of atrial natriuretic peptide); «mechanical» myocardial stress (soluble stimulating growth factor expressed by gene 2 — sST2), copeptin, galectin-3 in patients with heart failure and preserved LVEF, including older persons, as well as the possibility of their use in outpatient practice to predict the course of heart failure. The contribution of the multimarker model for a comprehensive assessment of prognosis is discussed, taking into account both the «hemodynamic» side of myocardial stress (pressure or volume overload, markers — natriuretic peptides), and «mechanical» (fibrosis / hypertrophy / heart remodeling, marker — sST2) myocardial changes.

Disruption of pathways regulated by Integrator complex in Galloway\u2013Mowat syndrome due to WDR73 mutations

Several studies have reported WDR73 mutations to be causative of Galloway–Mowat syndrome, a rare disorder characterised by the association of neurological defects and renal-glomerular disease. In this study, we demonstrate interaction of WDR73 with the INTS9 and INTS11 components of Integrator, a large multiprotein complex with various roles in RNA metabolism and transcriptional control. We implicate WDR73 in two Integrator-regulated cellular pathways; namely, the processing of uridylate-rich small nuclear RNAs (UsnRNA), and mediating the transcriptional response to epidermal growth factor stimulation. We also show that WDR73 suppression leads to altered expression of genes encoding cell cycle regulatory proteins. Altogether, our results suggest that a range of cellular pathways are perturbed by WDR73 loss-of-function, and support the consensus that proper regulation of UsnRNA maturation, transcription initiation and cell cycle control are all critical in maintaining the health of post-mitotic cells such as glomerular podocytes and neurons, and preventing degenerative disease.

Management of androgenetic alopecia: a review

<p class="abstract">Androgenetic alopecia is a patterned hair loss disorder affecting approximately 73% of men and 57% of women in their lifetime, this affection can cause psychological effects like low self-stem and depression diminishing the quality of life. Currently, finasteride and minoxidil are the only two drugs approved by the food and drug administration (FDA), and devices of low-level laser therapy are FDA cleared. Alternative therapies for androgenetic alopecia treatment include a drug like dutasteride, botulinum toxin A, cell-based therapies like adipose-derived stem cell-conditioned medium and platelet-rich plasma protocols. A combination of two or more therapies can be found in the literature, the general consent indicates that any combination has higher efficiency than a single therapy. Finasteride is in less use due to the related sexual side effects, some adjuvant techniques have emerged to improve the delivery and effectiveness of minoxidil, i.e. micro needling. The surgical approach is mainly focused on hair transplant surgery, which offers relatively less invasive procedures and is a more suitable option for long time results, however, it is expensive. Recently, drug-assisted delivery techniques have emerged to improve the efficiency of conventional drugs; transdermal drug delivery through ultrasound pressure waves means creating diffusion channels, increasing skin permeability and stimulating cell differentiation and growth factor utilization along with minoxidil absorption. Another approach for drug-assisted delivery is the embodiment of finasteride in polymer-based microspheres aiming for a sustained and controlled delivery, which can be beneficial to reduce the regular doses decreasing adverse effects.</p>

Platelet-Derived Growth Factor Stimulated Migration of Bone Marrow Mesenchymal Stem Cells into an Injectable Gelatin-Hydroxyphenyl Propionic Acid Matrix.

Bone marrow mesenchymal stem cells (bMSCs) are responsible in the repair of injured tissue through differentiation into multiple cell types and secretion of paracrine factors, and thus have a broad application profile in tissue engineering/regenerative medicine, especially for the musculoskeletal system. The lesion due to injury or disease may be a closed irregular-shaped cavity deep within tissue necessitating an injectable biomaterial permissive of host (endogenous) cell migration, proliferation and differentiation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) is a natural biopolymer hydrogel which is covalently cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in situ and can be delivered to the lesion by needle injection. Growth factors and cytokines can be directly incorporated into the gel or into nano- and micro-particles, which can be employed for sustained release of biomolecules while maintaining their bioactivity. In this study, we selected polyelectrolyte complex nanoparticles (PCNs) prepared with dextran sulfate and chitosan as the carrier for platelet-derived growth factor (PDGF)-BB and stromal cell-derived factor (SDF)-1α, which have been tested effectively in recruiting stem cells. Our in vitro results showed a high degree of viability of bMSCs through the process of Gtn-HPA covalent cross-linking gelation. The Gtn-HPA matrix was highly permissive of bMSC migration, proliferation, and differentiation. PDGF-BB (20 ng/mL) directly incorporated into the gel and, alternatively, released from PCNs stimulated bMSC migration and proliferation. There were only small differences in the results for the direct incorporation of PDGF into the gel compared with its release from PCNs, and for increased doses of the growth factor (200 ng/mL and 2 µg/mL). In contrast, SDF-1α elicited an increase in migration and proliferation only when released from PCNs; its effect on migration was notably less than PDGF-BB. The in vitro results demonstrate that PDGF-BB substantially increases migration of bMSCs into Gtn-HPA and their proliferation in the gel, and that these benefits can be derived from incorporation of a relatively low dose of the growth factor directly into the gel. These findings commend the use of Gtn-HPA/PDGF-BB as an injectable therapeutic agent to treat defects in musculoskeletal tissues.

Additional diagnostic criteria for atrial cardiomyopathy in patients with lone atrial fibrillation

Aim . To determine additional diagnostic criteria for class I atrial cardiomyopathy (ACM) in patients with lone atrial fibrillation (AF). Material and methods . This cross sectional non-randomized clinical study included 170 stable patients <60 years of age, of which 99 patients were selected. The inclusion criteria in the first group were the presence of lone AF with increased left atrial (LAVI) or right atrial volume index according to echocardiography without cardiovascular and pulmonary diseases, hypertension, and diagnostic criteria for heart failure (HF). The inclusion criterion in the second group was combination of AF and HF. The inclusion criterion in the third group was diagnostic criteria for HF (N-terminal pro-brain natriuretic peptide >125 ng/ml) in patients with sinus rhythm. In all patients, the concentration of NT-proBNP, a soluble stimulating growth factor 2 (sST2), as well as creatinine and cystatin C with calculation of the glomerular filtration rate, tissue inhibitor of matrix metalloproteinases-1 (TIMP1), and neutrophil gelatinase associated lipocalin (NGAL) was determined. Non-invasive angiography was performed. Results . According to ROC analysis, NT-proBNP, TIMP-1, NGAL as markers of class I ACM in patients with lone AF, showed unsatisfactory clinical significance. In patients with ACM and AF, regardless of the presence/ absence of HF, direct moderate relationship between sST2 and LAVI (r=0,470, p=0,012) and direct strong correlation between sST2 and NT-proBNP (r=0,726, p=0,004). Using ROC curve, for all available sST2 values, its diagnostic significance was obtained in the range from 5 to 16 ng/ml (AUC=0,98). Conclusion . The blood concentration of sST2 in the range from 5 to 16 ng/ml can be considered as an additional diagnostic criterion for class I ACM in patients with lone AF with a sensitivity of 98% and a specificity of 80%.

Abstract GS1-06: FGFR1 associates with gene promoters and regulates gene transcription: Implications for endocrine resistance in ER+/FGFR1-amplified breast cancer

Abstract Background: FGFR1 amplification occurs in ~ 15% of ER+ breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its role in transcriptional regulation is unclear. Thus, we investigated the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Results: FGFR1 ChIP-Seq detected 4,412 DNA binding sites in CAMA1 ER+/FGFR1-amplified breast cancer cells cultured in estrogen-free conditions. Of these binding sites, 67% were enriched at promoter regions. ChIP-qPCR confirmed FGFR1 binding to several promoter regions in a second ER+/FGFR1-amplified cell line, MDA-MB-134, and a patient derived xenograft, HCI-011. To determine the nuclear FGFR1 interactome, we performed FLAG immunoprecipitation of mixed nuclear and chromatin fractions of CAMA1 cells transduced with a 3XFLAG-FGFR1 plasmid, followed by mass spectrometry (MS) of FLAG antibody pulldowns. MS revealed RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P ChIP-Seq revealed that 2,867/4,412 (65%) FGFR1 peaks were shared with Pol II S5P. ChIP-Seq also showed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p<0.0001), suggesting that FGFR1 binds to actively transcribed genes. In addition to Pol II, MS detected FOXA1 among FGFR1 interacting proteins. ChIP-Seq analysis revealed FOXA1 enriched at FGFR1-bound loci. siRNA-mediated FOXA1 knockdown reduced FGFR1 distribution to several genomic loci in CAMA1 cells, as measured by FGFR1 ChIP-Seq, suggesting that FOXA1 mediates FGFR1 recruitment to chromatin. We next transduced MCF-7 cells with an FGFR1(SP-)(NLS) plasmid, where the NLS sequence forces nuclear import of the resulting protein. To determine the role of FGFR1 on transcriptional regulation, we used Binding and Expression Target Analysis (BETA), integrating FGFR1 ChIP-Seq and RNA-Seq results from MCF7FGFR1(SP-)(NLS) vs MCF7EV cells. This analysis predicted a direct role for genomic-bound FGFR1 in activating gene expression (p=8.01e-6). MCF7FGFR1(SP-)(NLS) cells were markedly less sensitive to fulvestrant compared to control cells. Gene Set Enrichment Analysis (GSEA) of the 1,009 genes upregulated in MCF7FGFR1(SP-)(NLS) cells and bound by FGFR1 at a genomic level revealed a strong enrichment of estrogen response early (q=2.2e-44) and late (q=6.4e-33) genes, suggesting that nuclear FGFR1 induces an ERα-associated transcriptional profile that may contribute to endocrine resistance. Finally, an expression signature associated with nuclear FGFR1 correlated with endocrine resistance in three cohorts of patients with ER+ breast cancer treated with aromatase inhibitors. We next studied the effect of growth factor stimulation on FGFR1 transcriptional function. Stimulation with FGF2 enhanced nuclear FGFR1 import in CAMA1 cells, as well as FGFR1-Pol II S5P association. Notably, these effects were not abrogated by treatment with the FGFR1 inhibitor erdafitinib. ChIP-Seq revealed that erdafitinib did not impair the FGFR1 genomic distribution. These results do not support a causal link between the FGFR1 activated TK and the receptor’s activity in the nucleus. Conclusions: We have demonstrated a role for nuclear FGFR1 in transcriptional regulation in breast cancer. FGFR1-induced gene expression contributes to endocrine resistance and is not affected by FGFR TKIs. These findings provide a rationale for developing treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1-amplified breast cancer. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Chang-Ching Lin, Kyung-min Lee, Dhivya R Sudhan, Ariella B Hanker, Sumanta Chatterjee, Angel Guerrero-Zotano, Paula Gonzalez-Ericsson, Saurabh Mendiratta, Hiroaki Akamatsu, Nicholas James, Ralf Kittler, Carlos L Arteaga. FGFR1 associates with gene promoters and regulates gene transcription: Implications for endocrine resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-06.