Hyaluronic Acid Levels Research Articles

HIF1α lactylation enhances KIAA1199 transcription to promote angiogenesis and vasculogenic mimicry in prostate cancer

Prostate cancer (PCa) is one of the most prevalent malignancies in adult males. However, PCa is resistant to multi-kinase inhibitors-based anti-angiogenic therapies, and the mechanism and effective targeting thereof remains unclear. In this study, single-cell and bulk-transcriptomic datasets analysis revealed that KIAA1199, a hyaluronic acid (HA) binding protein, was involved in glycolysis, hypoxia and angiogenesis pathways. Moreover, boosted KIAA1199 expression in PCa tissues was positively correlated with tumor stage, hypoxia-inducible factor (HIF)-1α overexpression, as well as angiogenesis markers. Tube formation, Western blot, enzyme-linked immunosorbent assay, and in vivo tumorigenesis results demonstrated that KIAA1199 silencing significantly inhibited angiogenesis and vasculogenic mimicry (VM), both in vitro and in vivo, by increasing semaphoring 3A (sema3A) expression while decreasing expressions of VEGFA, VE-cadherin, phosphorylated EphA2, and depolymerized HA levels. KIAA1199 overexpression was also found to promote angiogenesis and VM via increasing secretory VEGFA, however, this activity could be reversed by the HA biosynthesis inhibitor 4-methylumbelliferone (4MU). Furthermore, dual-luciferase and ChIP-PCR revealed that HIF1α is the transcriptional enhancer of KIAA1199, while lactate imported to PCa cells by monocarboxylate transporter 1 (MCT1) stabilizes HIF1α under normoxia via HIF1α lactylation. Our findings may provide a better understanding of angiogenesis and a promising therapeutic target of PCa.

ALL-548 Assessment of Serum Hyaluronic Acid and Ferritin Levels in Children With Acute Lymphoblastic Leukemia

Serum levels of hyaluronic acid (HA) and ferritin are high in newly diagnosed children with acute lymphoblastic leukemia. To evaluate the level of serum hyaluronic acid and ferritin in patients with ALL at diagnosis and in remission and investigate their impact on disease development and prognosis. A prospective cross-sectional study was conducted in the Central Teaching Hospital of Pediatrics, Baghdad, Iraq from January to October 2020. The study was conducted on hospitalized patients. For each patient, consent was obtained to take peripheral blood (PB) and/or bone marrow (BM) samples, and serum samples were analyzed in a private laboratory. 60 subjects were enrolled in this study, 40 ALL patients as cases (20 patients at diagnosis before starting chemotherapy and 20 patients in remission but still under therapy); and 20 age and sex-matched healthy subjects as controls. Both Band T-ALL subtypes were included. The age was less than 15 years. The patients were randomly collected in relation to gender. Patients on iron supplementation, with liver diseases, and those with other types of malignancy were excluded. The diagnosis of ALL was based on morphological assessments of the stained PB and BM smears according to the standard French-American-British criteria and confirmed by cytochemical stains and flow cytometric immunophenotyping. Patients with complete remission were also evaluated by morphological examination. Serum HA was measured using a commercially available sandwich ELISA kit while serum ferritin was measured by an immunoassay analyzer. Serum HA in newly diagnosed patients with ALL was significantly higher than either patients in remission (P-value 0.022) or controls (P-value 0.001). Both newly diagnosed patients and those in remission had elevated levels of serum ferritin with no significant difference. Serum levels of HA correlated positively with both serum ferritin and LDH (p<0.001). Our data suggest that serum level of hyaluronic acid could have a role in the malignant process of ALL and may serve as a possible marker for aggressiveness. Further studies on larger populations and on patients with relapse and follow-up of patients for a longer period of time are recommended.

Osteopontin (OPN) alleviates the progression of osteoarthritis by promoting the anabolism of chondrocytes

Osteoarthritis (OA) is a chronic debilitating joint disease, characterized by degeneration of the cartilage and loss of the cartilage matrix, and it is clinically manifested as joint pain. Osteopontin (OPN) is a glycoprotein that is abnormally expressed in the bone and cartilage tissues and plays a vital role in various pathological processes such as the osteoarthritic inflammatory response and endochondral ossification. The focus of our study is to investigate the therapeutic potential and specific role of OPN in OA. Using morphological comparisons, we found that the cartilage was severely worn-out and there was a significant loss of the cartilage matrix in OA. OPN, CD44, and hyaluronic acid (HA) synthase 1 (HAS1) were highly expressed, and the anabolism of HA was significantly higher in the OA chondrocytes than in the control chondrocytes. Additionally, we treated the OA chondrocytes with small interfering RNA (siRNA) targeting OPN, recombinant human OPN (rhOPN), and a combination of rhOPN and anti-CD44 antibodies. Furthermore, in vivo experiments were performed in mice. We found that OPN upregulated the expression of downstream HAS1 and increased the anabolism of HA through CD44 protein expression in OA mice compared with those in control mice. Moreover, intra-articular injection of OPN in mice with OA significantly inhibited OA progression. In summary, OPN initiates an intracellular cascade via CD44 which results in an anabolic increase in HA levels, thereby inhibiting OA progression. Therefore, OPN is a promising therapeutic agent in precision treatment of OA.

Crataegus sinaica defatted methanolic extract ameliorated monosodium iodoacetate-induced oxidative stress andinhibited inflammation in a rat model of osteoarthritis.

Osteoarthritis is a degenerative joint disease without definite treatment. It is characterized by intra-articular inflammation, cartilage degeneration, subchondral bone remodeling, and joint pain. The objective of the current study was to assess the anti-osteoarthritic effect and the possible underlying mechanism of action of Crataegus sinaica extract (CSE). Intra-articular injection of monosodium iodoacetate in the right knee joint of all rats was done except for the sham group. One week later, the anti-inflammatory efficacy of CSE (100, 200, 300 mg/kg, daily p.o) for 4 successive weeks versus ibuprofen (40 mg/kg, p.o) was assessed. Serum inflammatory cytokines; as well as weekly assessment of knee joint swelling, joint mobility, and motor coordination were done. At the end of the experiment, a histopathological investigation of the affected knee joints and an x-ray investigation were also executed. CSE significantly decreased joint swelling, pain behaviors, and serum levels of TNF-α, IL6, hyaluronic acid, and CTX-II. The radiographic findings revealed almost normal joint space with normal radiodensity and diameter in CSE-treated rats. As well, the histopathological and immunohistochemical investigations of the knee joints in CSE-treated groups retained the cartilage structure of knee joints. A significant reduction in the percentage of caspase-3-stained chondrocytes and a decrease in TGF-β1 immuno-positive areas in the synovial lining and sub lining were recorded in CSE-treated rats, compared to the osteoarthritis control group. This study approved the chondroprotective effects of CSE, and its ability to inhibit the pain associated with osteoarthritis.

Diagnostic value of HA, PC-III, IV-C, and LN in infants with congenital biliary atresia.

Hyaluronic acid (HA), type III procollagen III (PC-III), type IV collagen IV (IV-C), and laminin (LN) have certain diagnostic value for hepatobiliary diseases. No published studies have compared the diagnostic accuracy of these 4 indicators for the diagnosis of congenital biliary atresia (CBA) in infants. This study aimed to investigate the diagnostic value of HA, PC-III, IV-C, and LN in infants with CBA.From January 2017 to December 2020, 185 infants with nonphysiological jaundice in the Second Department of General Surgery at the Children’s Hospital of Hebei were enrolled in this study. Forty-six infants with CBA (CBA group) and 139 infants without CBA (noncongenital biliary atresia group) were diagnosed using ultrasonography, magnetic resonance imaging, intraoperative exploration, and intraoperative cholangiography. The levels of HA, PC-III, IV-C, and LN in the 2 groups were statistically analyzed. The diagnostic accuracy was determined using receiver operating characteristic curves and by calculating the area under the curve. Univariate and multivariate logistic regression analyses were performed to identify the independent risk factors.Compared to the noncongenital biliary atresia group, the levels of HA, PC-III, IV-C, and LN were significantly increased in the CBA group (P <.05). The receiver operating characteristic analysis showed the optimal cutoff values for HA, PC-III, IV-C, and LN were 162.7, 42.5, 199.7, and 101.2 ng/mL, and the area under the curves were 0.892, 0.762, 0.804, and 0.768, respectively. The sensitivity values for the diagnosis of CBA were 76.82%, 71.61%, 70.32%, and 72.28%, and the specificity values for the diagnosis of biliary atresia were 70.22%, 70.44%, 66.34%, and 68.71%, respectively. In the multivariate model, HA ≥162.7 ng/mL (odds ratio [OR] = 5.28, 95% confidence interval [CI]: 3.15–8.37), PC-III ≥42.5 ng/mL (OR = 4.61, 95% CI: 2.54–7.16), IV-C ≥199.7 ng/mL (OR = 5.02, 95% CI: 2.98–7.64), and LN ≥101.2 ng/mL (OR = 6.25, 95% CI: 2.41–10.07) remained associated with the occurrence of CBA.HA, PC-III, IV-C, and LN have high accuracy for the diagnosis of CBA in infants, and these factors are potential diagnostic biomarkers for CBA.

DETERMINATION OF HYALURONIC ACID AND N-PEPTIDE OF COLLAGEN TYPE III LEVELS IN SERUM AS PREDICTORS OF THE PERITONEAL ADHESIONS DEVELOPMENT IN CHILDREN

Introduction. Adhesive peritoneal disease (APD) is the most common and formidable complication of abdominalsurgery, which led to adhesive processing occurring in 70 to 90 % of operated patients. It is manifested by lateadhesion intestinal obstruction (LAIO), chronic abdominal pain, constipation and infertility.The aim of the study was to investigate the levels of hyaluronic acid (HA) and N-peptide collagen type III (N-PCIII) in serum to determine the course of APD in children.Material and methods. Serum levels of HA and N-PC type III were determined in 148 children aged 6-17 years (83boys and 65 girls). I group (main) – 38 patients with APD, LAIO treatment with the use solution of natrii hyaluronasand decamethoxine, II comparison group – 35 children with APD, LAIO treatment by traditional methods, III group – 45patients operated on for other acute surgical pathologies of GI tract in which after surgery for 5-10 years no signsof APD were noted. The control group consisted of 30 children operated on inguinal hernias.All parents were given informed voluntary written consent to conduct the research. Statistical processing of theobtained results was performed using Microsoft Office Excel and Statistica 10.0 (StatSoft Inc.).Results. An increase in HA level in the control, main and comparative groups on the 5th day of postoperative periodwas registered by 31,94, 126,96 and 39,60%, respectively, in comparison with the preoperative values; on the 14th daydecrease in HA level was observed in all groups, by 21,24, 32,79 and 25,83%, respectively, in comparison with the 5th day.Determination of the level of N-PC type III in all groups showed an increase in its number on the 5th day,compared with pre-surgery by 64.62, 57.40 and 79.32 %, respectively. On the 14th day the level was 2 times higher inthe main group and 2.86 times higher in the comparison group, compared with the control indicators. This indicatedthe stabilization of connective tissue organization and peritoneal regeneration in the main group and the lack of clearperitoneal regeneration in the comparison group.Conclusions.1. Serum levels of HA and N-PC type III may be markers of the onset and development of APD in children.2. An increase in the level of HA by more than 30%, and N-PC type III by 90 %, compared with control indicators,a year or more after undergoing surgery for APD, LAIO, may indicate a high risk of recurrence and be used as anadditional prognostic indicator.3. Intraoperative use of sodium hyaluronate and decamethoxine in abdominal surgery and the use of localadhesiolysis as anti-adhesive measures help reduce the risk of re-adhesion in children.

Hyaluronic acid restored protein permeability across injured human lung microvascular endothelial cells.

Lung endothelial permeability is a key pathological feature of acute respiratory distress syndrome. Hyaluronic acid (HA), a major component of the glycocalyx layer on the endothelium, is generated by HA synthase (HAS) during inflammation and injury and is critical for repair. We hypothesized that administration of exogenous high molecular weight (HMW) HA would restore protein permeability across human lung microvascular endothelial cells (HLMVEC) injured by an inflammatory insult via upregulation of HAS by binding to CD44. A transwell coculture system was used to study the effects of HA on protein permeability across HLMVEC injured by cytomix, a mixture of IL‐1β, TNFα, and IFNγ, with or without HMW or low molecular weight (LMW) HA. Coincubation with HMW HA, but not LMW HA, improved protein permeability following injury at 24 h. Fluorescence microscopy demonstrated that exogenous HMW HA partially prevented the increase in “actin stress fiber” formation. HMW HA also increased the synthesis of HAS2 mRNA expression and intracellular HMW HA levels in HLMVEC following injury. Pretreatment with an anti‐CD44 antibody or 4‐methylumbelliferone, a HAS inhibitor, blocked the therapeutic effects. In conclusion, exogenous HMW HA restored protein permeability across HLMVEC injured by an inflammatory insult in part through upregulation of HAS2.

Phloridzin Reveals New Treatment Strategies for Liver Fibrosis.

Liver fibrosis is an urgent public health problem which is difficult to resolve. However, various drugs for the treatment of liver fibrosis in clinical practice have their own problems during use. In this study, we used phloridzin to treat hepatic fibrosis in the CCl4-induced C57/BL6N mouse model, which was extracted from lychee core, a traditional Chinese medicine. The therapeutic effect was evaluated by biochemical index detections and ultrasound detection. Furthermore, in order to determine the mechanism of phloridzin in the treatment of liver fibrosis, we performed high-throughput sequencing of mRNA and lncRNA in different groups of liver tissues. The results showed that compared with the model group, the phloridzin-treated groups revealed a significant decrease in collagen deposition and decreased levels of serum alanine aminotransferase, aspartate aminotransferase, laminin, and hyaluronic acid. GO and KEGG pathway enrichment analysis of the differential mRNAs was performed and revealed that phloridzin mainly affects cell ferroptosis. Gene co-expression analysis showed that the target genes of lncRNA were obvious in cell components such as focal adhesions, intercellular adhesion, and cell–substrate junctions and in metabolic pathways such as carbon metabolism. These results showed that phloridizin can effectively treat liver fibrosis, and the mechanism may involve ferroptosis, carbon metabolism, and related changes in biomechanics.

Methods of a New Chronic Pancreatitis and Spontaneous Pancreatic Cancer Mouse Model Using Retrograde Pancreatic Duct Injection of Dibutyltin Dichloride.

The current study aimed to develop a new chronic pancreatitis and spontaneous pancreatic cancer model on C57/BL6 mouse through retrograde pancreatic duct injection of dibutyltin dichloride (DBTC) and explore its basic pathological changes as compared to the previous published chronic pancreatitis model through tail vein injection of DBTC with alcohol drinking. C57/BL6 mice were randomly divided into 3 groups: CG (control group; n = 15), VG (tail vein injection of DBTC (8 mg/kg) with 10% alcohol drinking group; n = 20), and PG (retrograde pancreatic duct injection of DBTC group (1 mg/kg); n = 30). Five mice in each group were sacrificed at a specific time point after the first treatment. The pathological section was observed. The activities of amylase, bilirubin, and hyaluronic acid in serum were determined. The expression of fibronectin, COL1A1, α-SMA, MMP-1, and TIMP-1 in the pancreas was assayed. Severe fibrosis of the pancreas with inflammatory cell infiltration could be observed on day 21 in the PG. In the VG, slight fibrosis of the pancreas with inflammatory cell infiltration was observed on day 28. There were significant differences in serum amylase, bilirubin, and hyaluronic acid levels between the PG and VG. The protein level of COL1A1 and α-SMA significantly increased in the PG. The mRNA expression of TIMP-1 is upregulated and the MMP-1 mRNA level is downregulated in the PG. Finally, typical neoplastic pathological change is significantly obvious in the PG. In conclusion, we established and validated a new chronic pancreatitis (CP) and spontaneous pancreatic cancer mouse model through retrograde injection of DBTC into the pancreatic duct. Previously reported mouse model through tail vein injection of DBTC with alcohol drinking could not cause obvious CP and neoplastic pathological change in mice.

Skin Dryness Induced in the KK-Ay/TaJcl Type 2 Diabetes Mouse Model Deteriorates Following Dapagliflozin Administration.

Various diabetic drugs have been developed as the number of patients with type 2 diabetes has increased. Sodium-glucose cotransporter (SGLT)-2 inhibitors have been developed as novel therapeutic agents. However, SGLT-2 inhibitors cause skin dryness. The mechanism through which SGLT-2 inhibitors cause skin dryness is unknown. The purpose of this study was to investigate the mechanism through which dapagliflozin, a SGLT-2 inhibitor, induces skin dryness. Specific pathogen-free KK-Ay/TaJcl (type 2 diabetes model) mice were orally administered with SGLT-2 inhibitor (dapagliflozin) daily for 4 weeks at a dose of 1 mg/kg/d. Skin dryness induced in KK-Ay/TaJcl mice became severe after dapagliflozin administration. Dapagliflozin treatment decreased collagen type I and hyaluronic acid levels in mice; additionally, it affected the transforming growth factor (TGF)-β/hyaluronan synthase pathway, further reducing hyaluronic acid levels. The results indicate that the reduction in hyaluronic acid levels plays an important role in the occurrence of dry skin in diabetes.

Extracts of Periplaneta americana alleviate hepatic fibrosis by affecting hepatic TGF-β and NF-κB expression in rats with pig serum-induced liver fibrosis.

Liver fibrosis is caused by continuous wound healing responses to various harmful stimuli, including viral infection, drugs, alcohol, and autoimmune liver disease. The purpose of this study was to examine the effects of extracts of Periplaneta americana (EPA) in rats with pig serum-induced liver fibrosis to preliminarily assess the antifibrotic effect of EPA. Seventy rats were randomly divided into 7 groups (10 rats in each group): HC, the healthy control group; FC, the fibrotic control group; TL, low-dose EPA treatment group group; TM, medium-dose EPA group; TH, high-dose EPA treatment group; TC1, Panax notoginseng/Salvia mitiorrhiza treatment control group 1; TC2, colchicine treatment control group 2. TC1 and TC2 were used as the positive control to demonstrate the difference between EPA and the effects of other compounds. The liver fibrosis model was induced by intraperitoneal injection of 0.5 mL pig serum twice a week for 13 weeks in all groups except for the HC group. The hepatic fibrosis model was established at the 7th week, and followingly, the corresponding compounds were administered once a day in all groups for 6 weeks. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity was determined in rat blood serum. We also measured liver fibrosis-related serum markers, including hyaluronic acid (HA), mucin layer (LN), type III pre-collagen (PC-III) and type IV collagen (IV-C). Hematoxylin and eosin (H&E) and Masson stainings were used to assess liver morphology and determine the stage of fibrosis. Immunohistochemistry was used to detect the protein expression of NF-κB, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in rat liver tissue. Compared with that of the HC group, the liver tissue of the FC group presented obvious liver damage and collagen deposition. The serum levels of ALT, AST, HA, LN, PC-III and IV-C and the expression of NF-κB, α-SMA, TGF-β1 and TIMP-1 in the FC group were significantly higher than those in the HC group, the EPA treatment groups, the TC1 group and the TC2 group (P < 0.01). The levels of serum ALT, AST, HA, LN, PC-III and IV-C and the expression of α-SMA, NF-κB, TGF-β1 and TIMP-1 in the TL, TC1 and TC2 groups were significantly higher than those TM and TH groups (P < 0.05). EPA treatment significantly improved liver function, decreased collagen deposition and reversed the pathological changes related to liver fibrosis. We found that EPA could reduce liver inflammation, suppress liver cell degeneration and necrosis, and reduce the formation of liver fibrous tissue. Its mechanism might be associated with inhibiting the expression of TGF-β1, TIMP-1, NF-κB and α-SMA to block signal transduction pathways in the hepatic fibrosis process. Therefore, EPA, as a traditional Chinese medicine, might be potentially used to prevent and treat hepatic fibrosis in the future. However, further more experiments are necessary to verify its effectiveness and possible signaling pathways.

Protective Effects and Molecular Mechanism of Total Flavonoids from Lycium Barbarum Leaves on Photoaged Human Dermal Fibroblasts.

Objective To investigate the effects and corresponding mechanisms of total flavonoids (TFL) from Lycium barbarum leaves on photoaged human dermal fibroblasts (HDFs). Methods Crude TFL was extracted with 70% ethanol, and a Rutin standard curve was drawn using the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry method to calculate its yield and mass concentration. After that, the photoaging HDFs model was established by UVA combined with 8-MOP. CCK-8 was performed to assess the influence of TFL on the proliferation of HDFs and photoaging HDFs. β-galactosidase (SA-β-gal) staining and activity assays were performed to evaluate the activity of SA-β-gal and the rate of SA-β-gal-positive cells in HDFs cells. The level of skin ECM proteins and oxidative stress-related substances in HDFs cells of each group was determined by ELISA and biochemical detection, respectively. Apoptosis of HDFs in each group was assessed by flow cytometry. The expressions of MAPK signaling pathway-related proteins in HDFs were detected by western blot. Results The yield rate of TFL extracted by 70% ethanol was 41.9%, and its purity rate was 34.6%. TFL at 25, 50, and 100 μg/mL was able to greatly promote the proliferation of HDFs. A photoaged HDFs model was successfully constructed by combining UVA irradiation at 9 J/cm2 and 8-MOP at 50 mg/L. TFL treatment could significantly inhibit apoptosis, SA-β-gal-positive cell staining rate, SA-β-gal activity, lactate dehydrogenase (LDH) leakage, and malondialdehyde (MDA) content in photoaged HDFs. Further, TFL increased the proliferative activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, type I collagen (Col I), hydroxyproline (HYP), and hyaluronic acid (HA) level of photoaged HDFs in a dose-dependent manner. Additional experiments suggested that TFL played a protective role by downregulating MAPK signaling pathway activity in photoaged HDFs cells. Conclusion TFL could inhibit oxidative stress and apoptosis, promote cell proliferation and the level of ECM-related component proteins, and participate in antiphotoaging in a concentration-dependent manner. The protective role of TFL in photoaged HDFs might be related to its inhibition of MAPK signaling pathways.

Risk factors for Fontan-associated hepatocellular carcinoma.

AimsThe incidence of hepatocellular carcinoma (HCC) in patients with Fontan-associated liver disease (i.e., FALD-HCC) has increased over time. However, the risk factors for HCC development remain unclear. Here, we compared the levels of non-invasive markers to the survival rate of FALD-HCC patients.MethodsFrom 2003 to 2021, 154 patients (66 men, 42.9%) developed liver disease after undergoing Fontan procedures. HCC was diagnosed in 15 (9.7%) (8 men, 53.3%) at a median age of 34 years (range, 21–45 years). We compared FALD-HCC and non-HCC cases; we generated marker level cutoffs using receiver operating characteristic curves. We sought to identify risk factors for HCC and mortality.ResultsThe incidence of HCC was 4.9% in FALD patients within 20 years after the Fontan procedure. Compared with non-HCC patients, FALD-HCC patients exhibited higher incidences of polysplenia and esophageal varices. At the time of HCC development, the hyaluronic acid (HA) level (p = 0.04) and the fibrosis-4 index (p = 0.02) were significantly higher in FALD-HCC patients than in non-HCC patients; the total bilirubin (T-BIL) level (p = 0.07) and the model for end-stage liver disease score [excluding the international normalized ratio (MELD-XI)] (p = 0.06) tended to be higher in FALD-HCC patients. Within approximately 20 years of the Fontan procedure, 10 patients died (survival rate, 96.9%). Kaplan–Meier curve analysis indicated that patients with T-BIL levels ≥ 2.2 mg/dL, HA levels ≥ 55.5 ng/mL, and MELD-XI scores ≥ 18.7 were at high risk of HCC, a generally poor prognosis, and both polysplenia and esophageal varices. Multivariate Cox regression analyses indicated that the complication of polysplenia [Hazard ratio (HR): 10.915] and a higher MELD-XI score (HR: 1.148, both p < 0.01) were independent risk factors for FALD-HCC.ConclusionsThe complication of polysplenia and a MELD-XI score may predict HCC development and mortality in FALD patients.

Abstract 2387: The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells

Abstract Metabolic changes are commonly seen in cancer. Tumor cells present a high rate of glucose and glutamine uptake and part of these nutrients supplies the hexosamine biosynthesis pathway (HBP), whose exacerbation has been related to solid tumors progression, including colorectal cancer. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end product of HBP, serves as the donor for several enzymes involved in glycan biosynthesis. Aberrant glycosylation is known to affect cellular and molecular mechanisms related to the malignant phenotype. Beta 1,6-GlcNAc branches on N-glycans (products of the MGAT5 enzyme) are associated with greater stability of transmembrane glycoproteins, as glutamine and glucose transporters, and growth factor receptors. Furthermore, changes in the availability of UDP-GlcNAc can affect the levels of hyaluronic acid (HA) and O-GlcNAc-modified proteins. The interplay between different direct demands for UDP-GlcNAc is not well understood and may help to better understand tumor metabolism. Therefore, we intend to evaluate in colorectal cancer cells how changes in levels of branched beta1,6-GlcNAc N-glycans can affect both the hexosamine pathway and the different processes which demand UDP-GlcNAc (such as O-GlcNAcylation and hyaluronic acid synthesis). For this, two experimental strategies were performed. The first focused on the inhibition of complex N-glycans using swainsonine, and the second focused on the MGAT5 inactivation using CRISPR-Cas9. The levels of UDP-GlcNAc were also manipulated using DON, an inhibitor of the GFAT enzyme. Initially, it was found that both the pharmacological inhibition of complex N-glycan biosynthesis and the specific inactivation of MGAT5 lead to significantly reduced levels of hexosamine pathway intermediates, activated monosaccharides, and proteins modified by O-GlcNAc. Furthermore, it was also observed that MGAT5 inactivation enhances the expression of genes encoding HA synthases (HAS2 and HAS3). Upon detection of metabolites by LC-MS, a multivariate analysis was performed using the statistical method of Partial Least-Squares Discriminant Analysis (PLS-DA), and it was observed that KO MGAT5 cells formed a metabolically distinct population from MOCK cells. Interestingly, after pharmacological inhibition of GFAT, an increase in the levels of beta 1,6-GlcNAc branched N-glycans and a reduction in O-GlcNAcylation were observed. Treatment with DON was also able to reduce HAS2 expression however an increased in HAS3 expression was observed. These data suggest that there is a relationship among different demands for UDP-GlcNAc in tumor cells and a possible regulatory role played by beta 1,6-GlcNAc branched N-glycans in this issue. This work contributes to a better understanding of interconnections between biochemical processes that demand UDP-GlcNAc in the context of colorectal cancer. Citation Format: Érika Elias Ferreira, Isadora de Araújo Oliveira, Adriane Regina Todeschini, Julio Cesar de Freitas-Junior. The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2387.

Clinical Significance of Serum Collagen Type IV and Procollagen Type III N-Peptide Levels in Diagnosis and Differential Diagnosis of Lymphedema.

Background: Lymphatic endothelial cells production or modification were closely related to the extracellular matrix (ECM) molecules. The serum hyaluronic acid (HA), laminin (LN), procollagen type III N-peptide (PIIINP), and collagen type IV (CGIV) levels were researched to explore the clinical significance of serum ECM proteins in the diagnosis and differentiation of lymphedema. Methods: Fifty-five patients were enrolled. They were divided into primary lymphedema (PLE), secondary lymphedema (SLE), and venous edema (VE) groups. Twenty-two healthy controls were also recruited as normal control (NC). Serum HA, LN, PIIINP, and CGIV levels of all subjects were assessed using chemiluminescence immunoassay. Statistical analysis and receiver operating characteristic (ROC) curves were used to data analysis. Results: The serum levels of CGIV were significantly decreased in both PLE and SLE groups compared with those in the NC group. Reduced serum CGIV levels were associated with the severity of lymphedema. The serum levels of CGIV and PIIINP were identified decreased in both PLE and SLE groups compared with those in the VE group. However, the levels of serum HA and LN were not observed significantly changed in both PLE and SLE groups than those in NC or VE group. Furthermore, ROC curve indicated that serum CGIV and PIIINP were capable of providing good diagnostic and differential diagnostic efficacy at the most appropriate cutoff point value. Conclusion: The serum levels of CGIV may have clinical significance in the diagnosis of lymphedema. CGIV and PIIINP may play a role in the differentiation of lymphedema from VE.

Higher Levels of Plasma Hyaluronic Acid and N-terminal Propeptide of Type III Procollagen Are Associated With Lower Kidney Function in Children With Non-alcoholic Fatty Liver Disease.

ObjectiveHyaluronic acid (HA) and N-terminal propeptide of type III procollagen (PIIINP) are two non-invasive biomarkers of liver fibrosis in non-alcoholic fatty liver disease (NAFLD). We examined the relationships of plasma levels of HA and PIIINP with kidney function in children with NAFLD.MethodsPlasma HA and PIIINP levels were measured using two commercially available enzyme-linked immunosorbent assay kits in a cohort of 106 Caucasian overweight or obese children with biopsy-proven NAFLD. Glomerular filtration rate (eGFR) was estimated using the Bedside Schwartz equation. Genotyping for the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 variant was performed using an allelic discrimination assay.ResultsChildren with fibrosis F2 had significantly higher plasma PIIINP and HA levels than those with F0 or F1 fibrosis. Liver fibrosis was positively associated with plasma HA and PIIINP, as well as with the presence of the risk allele G of PNPLA3 rs738409 variant, and negatively with eGFR. Moreover, eGFR showed significant inverse associations with HA and PIIINP levels, as well as the presence of G of PNPLA3 rs738409, and liver fibrosis stage. Notably, our multivariable regression models showed that higher plasma PIIINP (standardized beta coefficient: −0.206, P = 0.011) and HA levels (standardized beta coefficient: −0.531, P < 0.0001) were associated with lower eGFR values, even after adjustment for age, sex, systolic blood pressure, PNPLA3 rs738409 genotype, and any stage of liver fibrosis.ConclusionsHigher levels of HA and PIIINP were associated with lower eGFR values in Caucasian children with biopsy-proven NAFLD, independently of PNPLA3 rs738409 genotype and other potential confounding factors.

Hyaluronic acid detection and relative quantification by mass spectrometry imaging in human skin tissues.

Hyaluronic acid (HA) is a major component of the skin, contributing to tissue hydration and biomechanical properties. As HA content in the skin decreases with age, formulas containing HA are widely used in cosmetics and HA injections in aesthetic procedures to reduce the signs of aging. To prove the beneficial effects of these treatments, efficient quantification of HA levels in the skin is necessary, but remains difficult. A new analytical method has been developed based on matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify HA content in cross sections of human skin explants. A standardized and reproducible chemical entity (3 dimeric motifs or 6-mer) quantifiable by MALDI-MSI was produced by enzymatic hydrolysis using a specific hyaluronidase (H1136) in HA solution. This enzymatic digestion was carried out on skin sections before laser desorption, enabling the detection of HA. Histological coloration allowed us to localize the epidermis and the dermis on skin sections and, by comparison with the MALDI molecular image, to calculate the relative HA concentrations in these tissue areas. Skin explants were treated topically using a formula containing HA or its placebo, and the HA distribution profiles were compared with those obtained from untreated explants. A significant increase in HA was shown in each skin layer following topical application of the formula containing HA versus placebo and untreated samples (average of 126±40% and 92±40%, respectively). The MALDI-MSI technique enabled the quantification and localization of all HA macromolecules (endogenous and exogenous) on skin sections and could be useful for determining the efficacy of new cosmetic products designed to fight the signs of aging.

Development of skin health test efficacy modules in vivo

Sunlight contains ultraviolet (UV) light that causes sunburn and makes the skin age faster, leading to more wrinkles as older. The UV light can come from the natural and artificial sources. Moreover, UV light has shorter wavelengths than the visible light. Therefore, people’ eyes can’t see UV, but people’ skin can feel it. In this study, the in vivo skin health test efficacy modules have been established via the detection of skin’s moisture retention (%), skin’s cytokine expression levels, enzymatic expressions in the skin, the expression levels of hyaluronic acid (HA), collagen type I, melanin, and malondialdehyde (MDA) in the skin, and the experimental mice’ skin thickness and lesions via histo-pathologic examination. According to the results, the clinical behavior observation indexes of Institute of Cancer Research (ICR) mice in each group were normal during the experiments. Moreover, all ICR mice were survival until the end of the experiments. The moisture retention (%) of skin in ICR mice in UVB group was significant decrease after D1, D3, and D5 of UVB irradiation compared to the normal control group. Based on the IL-1β, IL-6 and TNF-α analysis expressions, both IL-1β and IL-6 expressions in UVB group were significantly increase than the control group, while there was no significant difference in the TNF-α expression between the groups. ICR mice’ skin enzymatic expressions in each group presented that catalase (CAT) expression and superoxide dismutase (SOD) activity in UVB group were significantly lower than the control group. The MDA expression in UVB group were significantly higher than the control group. The HA and collagen type I expressions in UVB group were significantly lower than the control group. However, the melanin expressions in UVB group and the control groups were not significantly different. The matrix metalloproteinase 2 (MMP-2) expressions in UVB group was significantly higher than the control group. The skin epidermal thickness in UVB group was significantly thicker than the control group. The dermal thickness in two groups was not significantly different. The number of sunburn cells in the derma in UVB group was significantly increase than the control group. The solar elastosis in the derma in two groups was not significantly different. Based on the above results, we have successfully established in vivo skin health test efficacy modules to evaluate the status of skin health. We hope the modules should be provide for the research and development (R&amp;D) of the effective treatment included drugs and therapeutic strategies.

Oxidation-mediated scaffold engineering of hyaluronic acid-based microcarriers enhances corneal stromal regeneration

The functional design of scaffolding biomaterials with potent capabilities of promoting cell adhesion and proliferation is critically important for tissue repair and regeneration. Here, we exploit the effects of oxidation level of aldehyde hyaluronic acid (oHA) on gelatin microcarriers for repairing corneal injuries. Specifically, high oxidation levels can endow the microcarrier surface with large oHA grafting amount, smooth topography, and strong stiffness, consequently formulating biocompatible scaffolding materials with superior affinities for keratocyte attachment and growth. In a rabbit model of corneal alkali burn injury, single intracorneal injection of keratocytes/functionalized microcarriers with an appropriate oxidation level could effectively reduce corneal swelling (~62-fold improvement), recover ~94% collagen production and ~89% keratocan expression, and repair disordered collagenous stromal architecture after 4 weeks. These findings on the oxidation level effects of the aldehyde polysaccharide show a great potential use in the development of advanced scaffolds for efficient tissue engineering.

In Vitro Anti-Photoaging and Skin Protective Effects of Licania macrocarpa Cuatrec Methanol Extract.

The Licania genus has been used in the treatment of dysentery, diabetes, inflammation, and diarrhea in South America. Of these plants, the strong anti-inflammatory activity of Licania macrocarpa Cuatrec (Chrysobalanaceae) has been reported previously. However, the beneficial activities of this plant on skin health have remained unclear. This study explores the protective activity of a methanol extract (50–100 μg/mL) in the aerial parts of L. macrocarpa Cuatrec (Lm-ME) and its mechanism, in terms of its moisturizing/hydration factors, skin wrinkles, UV radiation-induced cell damage, and radical generation (using RT/real-time PCR, carbazole assays, flowcytometry, DPPH/ABTS, and immunoblotting analysis). The anti-pigmentation role of Lm-ME was also tested by measuring levels of melanin, melanogenesis-related genes, and pigmentation-regulatory proteins. Lm-ME decreased UVB-irradiated death in HaCaT cells by suppressing apoptosis and inhibited matrix metalloproteinases 1/2 (MMP1/2) expression by enhancing the activity of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. It was confirmed that Lm-ME displayed strong antioxidative activity. Lm-ME upregulated the expression of hyaluronan synthases-2/3 (HAS-2/3) and transglutaminase-1 (TGM-1), as well as secreted levels of hyaluronic acid (HA) via p38 and JNK activation. This extract also significantly inhibited the production of hyaluronidase (Hyal)-1, -2, and -4. Lm-ME reduced the melanin expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1/2 (TYRP-1/2) in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 cells via the reduction of cAMP response element-binding protein (CREB) and p38 activation. These results suggest that Lm-ME plays a role in skin protection through antioxidative, moisturizing, cytoprotective, and skin-lightening properties, and may become a new and promising cosmetic product beneficial for the skin.