Combination of Fushengong decoction with Western medicine on patients with chronic renal failure: An observational study.

Chronic renal failure (CRF) causes a reduction in glomerular filtration rate and damage to renal parenchyma. Fushengong decoction (FSGD) showed improvement in renal function in CRF rats. This study aims to analyze the differentially expressed proteins in CRF patients treated with Western medicine alone or in combination with FSGD. Sixty patients with CRF recruited from Yongchuan Traditional Chinese Medicine Hospital affiliated to Chongqing Medical University were randomly assigned into control (treated with Western medicine alone) and observation groups (received additional FSGD treatment thrice daily for 8 weeks). The clinical efficacy and changes in serum Bun, serum creatinine, Cystatin C, and transforming growth factor beta 1 (TGF-ß1) before and after treatment were observed. We employed isotope relative labeling absolute quantification labeling and liquid chromatography-mass spectrometry to identify differentially expressed proteins and carried out bioinformatics Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Patients in the observation group showed greater clinical improvement and lower levels of serum Bun, serum creatinine, Cyc-c, and TGF-ß1 than the control group. We identified 32 differentially up-regulated and 52 down-regulated proteins in the observation group. These proteins are involved in the blood coagulation system, protein serine/threonine kinase activity, and TGF-ß, which are closely related to the pathogenesis of CRF. Protein-protein-interaction network analysis indicated that candidate proteins fibronectin 1, fibrinogen alpha chain, vitronectin, and Serpin Family C Member 1 were in the key nodes. This study provided an experimental basis suggesting that FSGD combined with Western medicine could significantly improve renal function and renal fibrosis of CRF patients, which may be through the regulation of fibronectin 1, fibrinogen alpha chain, vitronectin, Serpin Family C Member 1, TGF-ß, and the complement coagulation pathway (see Graphical abstract S1, Supplemental Digital Content, http://links.lww.com/MD/L947).

Intracellular Zn2+ promotes extracellular matrix remodeling in dexamethasone-treated trabecular meshwork.

Given the widespread application of glucocorticoids in ophthalmology, the associated elevation of intraocular pressure (IOP) has long been a vexing concern for clinicians, yet the underlying mechanisms remain inconclusive. Much of the discussion focuses on the extracellular matrix (ECM) of trabecular meshwork (TM). It is widely agreed that glucocorticoids impact the expression of matrix metalloproteinases (MMPs), leading to ECM deposition. Since Zn2+ is vital for MMPs, we explored its role in ECM alterations induced by dexamethasone (DEX). Our study revealed that in human TM cells treated with DEX, the level of intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. This correlated with changes in several Zrt-, Irt-related proteins (ZIPs) and metallothionein. ZIP8 knockdown impaired extracellular Zn2+ uptake, but Zn2+ chelation did not affect ZIP8 expression. Resembling DEX's effects, chelation of Zn2+ decreased MMP2 expression, increased the deposition of ECM proteins, and induced structural disarray of ECM. Conversely, supplementation of exogenous Zn2+ in DEX-treated cells ameliorated these outcomes. Notably, dietary zinc supplementation in mice significantly reduced DEX-induced IOP elevation and collagen content in TM, thereby rescuing the visual function of the mice. These findings underscore zinc's pivotal role in ECM regulation, providing a novel perspective on the pathogenesis of glaucoma.NEW & NOTEWORTHY Our study explores zinc's pivotal role in mitigating extracellular matrix dysregulation in the trabecular meshwork and glucocorticoid-induced ocular hypertension. We found that in human trabecular meshwork cells treated with dexamethasone, intracellular Zn2+ significantly decreased, accompanied by impaired extracellular Zn2+ uptake. Zinc supplementation rescues visual function by modulating extracellular matrix proteins and lowering intraocular pressure, offering a direction for further exploration in glaucoma management.

FGF23 directly inhibits osteoprogenitor differentiation in Dmp1-knockout mice.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1-knockout (Dmp1KO) mouse model of ARHR. At 12 weeks, Dmp1KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors' differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.

Ecliptasaponin A attenuates renal fibrosis by regulating the extracellular matrix of renal tubular cells.

Renal fibrosis is the most common manifestation of end-stage renal disease (ESRD), including diabetic kidney disease (DKD), but there is no effective treatment in renal fibrosis. Natural products are a rich source of clinical drug research and have been used in the clinical research of various diseases. In this study, we searched for traditional Chinese medicine monomers that attenuate fibrosis and assessed their effect on the fibrosis marker connective tissue growth factor (CTGF) in cells which we found ecliptasaponin A. Subsequently, we evaluated the effect of ecliptasaponin A on renal fibrosis in the classic renal fibrosis unilateral ureteral obstruction (UUO) mouse model and found that ecliptasaponin A could reduce the renal collagen fiber deposition and renal extracellular matrix (ECM) protein expression in UUO mice. In vitro, ecliptasaponin A can inhibit ECM protein expression in human kidney-2 (HK-2) cells induced by transforming growth factor-beta1 (TGFß1). To further clarify the mechanism of ecliptasaponin A in attenuating renal fibrosis, we performed transcriptome sequencing of HK-2 cells treated with TGFß1 and ecliptasaponin A. The functions and pathways were mainly enriched in the extracellular matrix and TGFß signalling pathway. Matrix metalloproteinase 10 (MMP10) and matrix metalloproteinase 13 (MMP13) are the main differentially expressed genes in extracellular matrix regulation. Then, we measured MMP10 and MMP13 in the cells and found that ecliptasaponin A had a significant inhibitory effect on MMP13 expression but not on MMP10 expression. Furthermore, we overexpressed MMP13 in HK-2 cells treated with TGFß1 and found that MMP13 promoted HK-2 cell injury. Our findings suggest that ecliptasaponin A can attenuate renal fibrosis, which may provide a new method for treating renal fibrosis clinically.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway.

BACKGROUND: Liver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PURPOSE: This study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. METHODS: The anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. RESULTS: The invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. CONCLUSION: This study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Long non-coding RNA H19 regulates matrisome signature and impacts cell behavior on MSC-engineered extracellular matrices.

BACKGROUND: The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. METHODS: Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. RESULTS: Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. CONCLUSION: Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.

Osteocytes and the pathogenesis of hypophosphatemic rickets.

Since phosphorus is a component of hydroxyapatite, its prolonged deprivation affects bone mineralization. Fibroblast growth factor 23 (FGF23) is essential for maintaining phosphate homeostasis and is mainly produced by osteocytes. FGF23 increases the excretion of inorganic phosphate (Pi) and decreases the production of 1,25-dihydroxyvitamin D in the kidneys. Osteocytes are cells of osteoblastic lineage that have undergone terminal differentiation and become embedded in mineralized bone matrix. Osteocytes express FGF23 and other multiple genes responsible for hereditary hypophosphatemic rickets, which include phosphate-regulating gene homologous to endopeptidase on X chromosome (PHEX), dentin matrix protein 1 (DMP1), and family with sequence similarity 20, member C (FAM20C). Since inactivating mutations in PHEX, DMP1, and FAM20C boost the production of FGF23, these molecules might be considered as local negative regulators of FGF23. Mouse studies have suggested that enhanced FGF receptor (FGFR) signaling is involved in the overproduction of FGF23 in PHEX-deficient X-linked hypophosphatemic rickets (XLH) and DMP1-deficient autosomal recessive hypophosphatemic rickets type 1. Since FGFR is involved in the transduction of signals evoked by extracellular Pi, Pi sensing in osteocytes may be abnormal in these diseases. Serum levels of sclerostin, an inhibitor Wnt/ß-catenin signaling secreted by osteocytes, are increased in XLH patients, and mouse studies have suggested the potential of inhibiting sclerostin as a new therapeutic option for the disease. The elucidation of complex abnormalities in the osteocytes of FGF23-related hypophosphatemic diseases will provide a more detailed understanding of their pathogenesis and more effective treatments.

Procollagen C-proteinase enhancer-1 and renal failure in multiple myeloma.

BACKGROUND: Renal involvement is present in approximately 50% of multiple myeloma (MM) cases and is associated with a poor prognosis. Procollagen C-Proteinase Enhancer 1 (PCPE-1) is an extracellular matrix glycoprotein that has been shown to increase collagen production by enhancing the activity of Procollagen C-Proteinase (PCP) involved in collagen fibrillogenesis and contribute to the fibrotic process. This study investigates the relationship between PCPE-1 and renal function in myeloma patients. METHODS: Eighty-one adults, consisting of 61 patients diagnosed with MM and 20 healthy controls, were included in this cross-sectional study. The MM patients with renal injury (RI) were classified as "MM-RI( +)" and those with no RI as "MM-RI(-)". RESULTS: The median serum PCPE-1 level was 10.7 (5.0-39.4) ng/mL for the entire study population, 9.9 (5.0-13.6) ng/mL for the control group, 10.0 (6.4-22.5) ng/mL for the MM-RI(-) group, and 11.4 (8.1-39.4) ng/mL for the MM-RI( +) group. The difference between the control group and MM-RI( +) group was statistically significant (p < 0.013). PCPE-1 levels negatively correlated with estimated glomerular filtration rate (eGFR), serum albumin, and hemoglobin levels but positively correlated with serum creatinine and CRP levels in the entire study population. Among MM patients, only serum phosphorus and beta-2-microglobulin (ß2M) were positively correlated with PCPE-1. PCPE-1 levels was not affected by other parameters in the entire study population and in the MM group. CONCLUSIONS: Although serum PCPE-1 was higher in the MM-RI( +) group, it was thought to be associated with low GFR reflecting non-specific kidney injury rather than myeloma-related kidney injury.

VItamin K In PEritonial DIAlysis (VIKIPEDIA): Rationale and study protocol for a randomized controlled trial.

Vascular calcification (VC) is an active process, resulting from the disturbance of balance between inhibitors and promoters of calcification, in favor of the latter. Matrix Gla Protein, a powerful inhibitor of VC, needs vitamin K to become active. In vitamin K depletion, plasma levels of the inactive form of MGP, dephosphorylated, uncarboxylated MGP (dp-ucMGP) are increased and associated with VC and cardiovascular (CV) outcomes. End Stage Renal Disease (ESRD) patients have increased circulating dp-ucMGP levels and accelerated VC. VItamin K In PEritoneal DIAlysis (VIKIPEDIA) is a prospective, randomized, open label, placebo-controlled trial, evaluating the effect of vitamin K2 supplementation on arterial stiffness and CV events in ESRD patients undergoing peritoneal dialysis (PD). Forty-four PD patients will be included in the study. At baseline, dp-ucMGP and pulse-wave velocity (PWV) will be assessed and then patients will be randomized (1:1 ratio) to vitamin K (1000 µg MK-7/day) or placebo for 1.5 years. The primary endpoint of this trial is the change in PWV in the placebo group as compared to the treatment group. Secondary endpoints are the occurrence of CV events, mortality, changes in PD adequacy, change in 24-hour ambulatory blood pressure indexes and aortic systolic blood pressure and changes in calcium/phosphorus/parathormone metabolism. VIKIPEDIA is a new superiority randomized, open label, placebo-controlled trial aiming to determine the effect of vitamin K2 supplementation on VC, CV disease and calcium/phosphorus metabolism, in PD patients. Trial registration: The protocol of this study is registered at ClinicalTrials.gov with identification number NCT04900610 (25 May 2021).

Arctigenin inhibits apoptosis, extracellular matrix degradation, and inflammation in human nucleus pulposus cells by up-regulating miR-483-3p.

BACKGROUND: Arctigenin (ATG) is the active ingredient of the Chinese herbal medicine Arctium lappa, with anti-inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are important causes of intervertebral disc degeneration (IDD). Hence, this study probed into the possible role of ATG in IDD. METHODS: Interleukin (IL)-1ß (10 ng/ml) was adopted to induce human nucleus pulposus cells (HNPCs) as a cell model for IDD. The effects of different concentrations of ATG (0, 2, 5, 10, 20, 50 µmol/L) on the viability of HNPCs and effects of ATG (10, 50 µmol/L) on the viability of IL-1ß-induced HNPCs were detected by cell counting kit-8 (CCK-8). After IL-1ß-induced HNPCs were transfected with miR-483-3p inhibitor and/or treated with ATG, cell viability and apoptosis were determined by CCK-8 and flow cytometry; the expressions of miR-483-3p, extracellular matrix (ECM)-related genes, and inflammation-related genes were measured by quantitative real time polymerase chain reaction (qRT-PCR), and expressions of ECM/apoptosis/NF-κB pathway-related proteins were quantified by Western blot. RESULTS: ATG had no significant effect on the viability of HNPCs but could promote the viability of IL-1ß-induced HNPCs. ATG inhibited apoptosis, ECM degradation, inflammation, and activation of NF-κB pathway in HNPCs induced by IL-1ß, but promoted the expression of miR-483-3p. MiR-483-3p inhibitor reversed the above-mentioned regulatory effects of ATG. CONCLUSION: Arctigenin suppresses apoptosis, ECM degradation, inflammation, and NF-κB pathway activation in HNPCs by up-regulating miR-483-3p.

Ginsenoside Rb1 prevents osteoporosis via the AHR/PRELP/NF-κB signaling axis.

BACKGROUND: Accumulating clinical and experimental evidence shows multiple biological effects of ginsenoside Rb1 (GRb1) in the treatment of aging related diseases such as osteoporosis (OP). Recently, GRb1 has attracted extensive attention as an anti-osteoporosis agent. Here, we sought to identify the mechanism by which GRb1 improves OP. METHODS: A dexamethasone (DEX)-induced rat model of OP was constructed and the rats were treated with GRb1 to examine its role in OP. We screened the action targets of GRb1 online and validated by performing functional experiments. The correlation between aryl hydrocarbon receptor (AHR) and proline/arginine-rich end leucine-rich repeat protein (PRELP) was identified through luciferase and chromatin immunoprecipitation assays. In the isolated osteoblasts from DEX-induced OP rats, the expression of osteogenic differentiation-associated genes, and nuclear factor-kappa B (NF-κB) pathway-related genes, mineralization, and number of calcium nodules were assessed. RESULTS: GRb1 enhanced the differentiation of osteoblasts, the mechanism of which was related to upregulation of AHR. AHR could promote the transcription of PRELP by binding to the PRELP promoter region and consequently caused its upregulation. Meanwhile, PRELP inhibited the activation of the NF-κB pathway, which underlay the promoting impact of AHR in the osteogenic differentiation. Additionally, GRb1 could ameliorate OP in DEX-induced rats via the AHR/PRELP/NF-κB axis. CONCLUSIONS: Our findings demonstrate that GRb1 might function as an effective candidate to prevent the progression of OP via regulation of the AHR/PRELP/NF-κB axis, revealing a new molecular mechanism underpinning the impact of GRb1 in the progression of OP and offering a theoretical contribution to the treatment of OP.

Increased Dephospho-uncarboxylated Matrix Gla-Protein Is Associated With Lower Axial Skeletal Muscle Mass in Patients With Hypertension.

BACKGROUND: Matrix Gla-protein (MGP) is a well-established inhibitor of vascular calcification that is activated by vitamin K-dependent carboxylation. In the setting of vitamin K2 deficiency, dephospho-uncarboxylated MGP (dpucMGP) levels increase, and have been associated with large artery stiffening. Vitamin K2 is also a mitochondrial electron carrier in muscle, but the relationship of vitamin K2 deficiency and dpucMGP with muscle mass is not well understood. We therefore aimed to examine the association of vitamin K2 deficiency and dpucMGP with skeletal muscle mass in patients with hypertension. METHODS: We studied 155 hypertensive adults without heart failure. Axial skeletal muscle mass was measured using magnetic resonance imaging from axial steady-state free precession images. DpucMGP was measured with ELISA. Carotid-femoral pulse wave velocity (CF-PWV) was measured from high-fidelity arterial tonometry recordings. RESULTS: We found an inverse relationship between dpucMGP levels and axial muscle mass, with progressively rising dpucMGP levels correlating with decreasing axial muscle mass. In an unadjusted linear regression model, correlates of dpucMGP included axial skeletal muscle area factor (ß = -0.32; P < 0.0001) and CF-PWV (ß = 0.31; P = 0.0008). In adjusted analyses, independent correlates of dpucMGP included axial skeletal muscle area factor (ß = -0.30; P = 0.0003) and CF-PWV (ß = 0.20; P = 0.019). CONCLUSIONS: In hypertensive adults, dpucMGP is independently associated with lower axial muscle mass, in addition to increased large artery stiffness. Further studies are required to investigate the role of vitamin K supplementation in this population.

Clinical and laboratory characterization of patients with localized scleroderma and response to UVA-1 phototherapy: In vivo and in vitro skin models.

BACKGROUND/PURPOSE: Localized scleroderma (LS) is a rare disease leading to progressive hardening and induration of the skin and subcutaneous tissues. LS is responsive to UVA-1 phototherapy, though its exact mechanism of action dermal fibrosis is yet to be fully elucidated. We aimed to investigate the molecular changes induced by UVA-1 rays in human primary fibroblasts cultures. METHODS: A total of 16 LS patients were treated with medium-dose UVA-1 phototherapy. At baseline, during and after therapy, Localized Scleroderma Assessment Tool, Dermatology Life Quality Index and lesions' staging and mapping were performed along with high-frequency ultrasound (HFUS) examination for dermal thickness assessment. Gene expression analysis for 23 mRNA transcripts, in vitro UVA-1 irradiation and viability tests were realized on lesional fibroblasts' primary cultures, before and 3 months after therapy. RESULTS: The dermal thickness, the LoSCAT and the DLQI progressively decreased starting from the last phototherapy session up to the 6 and 9 month follow-ups (-57% and -60%, respectively). Molecular gene analysis (rt-PCR) revealed that UVA-1 phototherapy exerts multiple effects: the activation of specific anti-fibrotic pathways (e.g., overexpression of CTHRC1 and metalloproteases 1, 2, 7, 8, 9, 12, suppression of TIMP-1), the downregulation of peculiar pro-fibrotic pathways (e.g., downregulation of TGF-ß, TGF-ßrII, Grb2, SMAD 2/3, TNRSF12A, CTGF) through a significant overexpression of IL-1ß; the stabilization of collagen synthesis acting on genes COL1A1, COL3A1, COL8A1, COL10A1, COL12A1. CONCLUSION: UVA-1 phototherapy adds significant benefits in local tissue remodeling, rebalancing the alteration between pro-fibrotic and anti-fibrotic pathways; these changes can be well monitored by HFUS.

MGP Regulates Perivascular Adipose-Derived Stem Cells Differentiation Toward Smooth Muscle Cells Via BMP2/SMAD Pathway Enhancing Neointimal Formation.

Perivascular adipose-derived stem cells (PV-ADSCs) could differentiate into smooth muscle cells (SMCs), participating in vascular remodeling. However, its underlying mechanism is not well explored. Our previous single-cell RNA-sequencing dataset identified a unique expression of matrix Gla protein (MGP) in PV-ADSCs compared with subcutaneous ADSCs. MGP involves in regulating SMC behaviors in vascular calcification and atherosclerosis. In this study, we investigated MGP's role in PV-ADSCs differentiation toward SMCs in vitro and in vascular remodeling in vivo. PV-ADSCs were isolated from perivascular regions of mouse aortas. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence confirmed higher MGP expression in PV-ADSCs. The MGP secretion increased along PV-ADSCs differentiation toward SMCs in response to transforming growth factor-beta 1 (TGF-ß1). Lentivirus knockdown of MGP markedly promoted the bone morphogenetic protein 2 (BMP2) expression and phosphorylation of SMAD1/5/8 in PV-ADSCs, subsequently inhibiting its differentiation toward SMCs. Such inhibition could be partially reversed by further application of BMP2 inhibitors. On the contrary, exogenous MGP inhibited BMP2 expression and SMAD1/5/8 phosphorylation in PV-ADSCs, thereby promoting its differentiation toward SMCs. Transplantation of cultured PV-ADSCs, which was pretreated by MGP knockdown, in mouse femoral artery guide-wire injury model significantly alleviated neointimal hyperplasia. In conclusion, MGP promoted the differentiation of PV-ADSCs toward SMCs through BMP2/SMAD-mediated signaling pathway. This study offers a supplement to the society of perivascular tissues and PV-ADSCs.

Biological Role of Vitamin K-With Particular Emphasis on Cardiovascular and Renal Aspects.

Vitamin K (VK) plays many important functions in the body. The most important of them include the contribution in calcium homeostasis and anticoagulation. Vascular calcification (VC) is one of the most important mechanisms of renal pathology. The most potent inhibitor of this process-matrix Gla protein (MGP) is VK-dependent. Chronic kidney disease (CKD) patients, both non-dialysed and hemodialysed, often have VK deficiency. Elevated uncarboxylated matrix Gla protein (ucMGP) levels indirectly reflected VK deficiency and are associated with a higher risk of cardiovascular events in these patients. It has been suggested that VK intake may reduce the VC and related cardiovascular risk. Vitamin K intake has been suggested to reduce VC and the associated cardiovascular risk. The role and possibility of VK supplementation as well as the impact of anticoagulation therapy on VK deficiency in CKD patients is discussed.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation.

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

Recognition of potential therapeutic role of 2-hydroxy-3-methylanthraquinones in the treatment of gallbladder carcinoma: A proteomics analysis.

Gallbladder carcinoma (GBC), with early metastasis and high recurrence rates, is an enormous threat to health. As an anthraquinones monomer of traditional Chinese medicine Hedyotis diffusa, 2-hydroxy-3-methylanthraquinone (HMA) has been reported to inhibit the growth of several cancers. But in our preliminary study, HMA could only weakly induce GBC cell apoptosis. To explore other possible mechanism underlying the inhibition effect of HMA on GBC, this proteomics analysis was performed. A proteomics analysis was performed on one GBC cell line bought from the China Life Science Cell Bank. Several computational techniques were merged to develop analysis for those differently expressed proteins. A comparative protein-protein interaction network analysis was carried out among the differently expressed proteins to identify the proteins potentially inhibiting GBC. Thus, a GO and KEGG analysis was performed to identify the signaling pathways underlying a potential therapeutic role for HMA. A total of 285 proteins were affected by HMA, including 187 upregulated and 98 downregulated. The subcellular localization of differently expressed proteins were identified, including 142 in nuclear, 67 in cytoplasm, 67 in extracellular matrix, 46 in plasma membrane, 13 in mitochondrion, 3 in lysosome, and 1 in cytoskeleton. HMA could regulate EGFR, FN1, PLG, PLAUR, LAMA3, HRG, THBS1, PLAT, KNG1, ENAM, SERPINE1, ECM1, interleukin-8, and trypsin in GBC. Most of the regulated proteins involve in cell migration. Pathways including PI3K-Akt, Wnt, HIF-1, focal adhesion, microRNAs were regulated by HMA. HMA was shown to be an inhibition agent for GBC development, and this analysis would contribute to the development of new anti-GBC drugs.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.