Collagen XVIII Research Articles

Interaction between prostate cancer cells and prostate fibroblasts promotes accumulation and proteolytic processing of basement membrane proteins.

Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, β2, and β3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.

Endostatin Is an Independent Risk Factor of Graft Loss after Kidney Transplant

Background: Endostatin is a 20-kDa C-terminal fragment of collagen XVIII, known for its ability to inhibit the proliferation of capillary endothelial cells. Previous studies suggested that circulating endostatin independently predicts incident chronic kidney disease. However, the impact of endostatin on graft loss level in kidney transplant recipients (KTRs) remains unknown. Methods: We conducted a prospective observational cohort study in 574 maintenance KTRs. Patients were followed for kidney graft loss and all-cause mortality during a median follow-up of 48 months. Serum-, and urine-samples and clinical data were collected at baseline. Serum Endostatin concentration was analyzed by an ELISA. Results: Among 574 patients, 37 patients had graft loss and 62 patients died. For graft loss, the optimal cut-off value based on receiver operating characteristics analysis (area under the curve 0.79, 95% CI 0.71–0.86, p < 0.001) of endostatin was 147.3 pmol/L. Kaplan-Meier curves revealed that higher serum endostatin concentrations positively correlated with graft loss (p < 0.001). Multivariable Cox regression analyses showed that baseline endostatin concentrations were significantly associated with graft loss after adjusting for graft loss risk factors (adjusted hazard ratio [HR] 8.34; 95% CI 2.19–31.72; p = 0.002). The adjusted HRs for classical graft loss risk factors such as baseline estimated glomerular filtration rate and urinary protein excretion were lower (1.91 and 5.44, respectively). In contrast to graft loss, baseline endostatin concentrations were not associated with all-cause mortality. Conclusion: Increased serum endostatin at baseline is independently associated with the risk of graft loss in KTRs.

Effect of endostatin overexpression on angiotensin II-induced cardiac hypertrophy in rats.

Background:Endostatin, a biologically active fragment of collagen XVIII, has been observed in patients with ischemic heart disease. The aim of the present study was to investigate whether endostatin overexpression could attenuate cardiac hypertrophy by inhibiting the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling pathway.Methods:This study was examined in vivo in rats and in vitro in primary neonatal rat cardiomyocytes treated with angiotensin (Ang) II to model cardiac hypertrophy. Twenty-four male Sprague-Dawley rats were randomized into adenovirus (Ad)-green fluorescent protein, Ang II, Ad-endostatin, and Ang II + Ad-endostatin groups (n = 6 in each group). Four weeks later, all the rats were weighed and sacrificed after transthoracic echocardiography. Cardiac function was evaluated by transthoracic echocardiography, cardiomyocyte size was evaluated by hematoxylin-eosin staining. Levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were evaluated by quantitative reverse-transcription polymerase chain reaction or Western blotting, PKA level was evaluated by Western blotting, and cAMP level was evaluated by enzyme-linked immunosorbent assay. Statistical significance among multiple groups was evaluated by one-way analysis of variance.Results:Endostatin overexpression reduced the increases in left ventricle (LV) mass (P = 0.0063), LV mass/body weight (BW) (P = 0.0013), interventricular septal thickness (IVS) in diastole (P = 0.0013), IVS in systole (P = 0.0056), left ventricular posterior wall thickness (LVPW) in diastole (P = 0.0291), LVPW in systole (P = 0.0080), heart weight (HW) (P = 0.0138), HW/BW (P = 0.0001), and HW/tibial length (P = 0.0372) in Ang II-treated rats. In addition, endostatin overexpression reduced cardiomyocyte cross-sectional area expansion, and reduced the levels of ANP and BNP in Ang II-treated rats (P = 0.0251 and 0.0477 for messenger RNA [mRNA]), and primary neonatal rat cardiomyocytes (P = 0.0188 and P = 0.0024 for mRNA; P = 0.0023 and 0.0013 for protein, respectively). Additionally, endostatin overexpression reduced the increase of cAMP (P = 0.0054) and PKA (P = 0.0328) levels in cardiomyocytes treated with Ang II. Treatment with cAMP reversed the effects of endostatin overexpression on ANP (P = 0.0263) and BNP (P = 0.0322) levels in cardiomyocytes induced by Ang II.Conclusion:Endostatin overexpression could alleviate cardiac hypertrophy by inhibiting the cAMP-PKA signaling pathway.

Positive association between abdominal aortic diameter and serum collagen XVIII levels.

The identification of abdominal aortic aneurysm (AAA) biomarker offers a perspective to determine disease progress and rupture risk. The aim of our study was to evaluate the association between selected circulating biomarkers and diameter of abdominal aorta. One hundred and two patients (88 men and 14 women) with mean age 70.0±8.7 years were included in a single center cross-sectional study conducted between February 2016 and October, 2018. AAA was defined as subrenal aortic dilatation ≥3 cm. Serum biomarker concentrations (insulin-like growth factor-1, peroxiredoxin-1, collagen IV, collagen XVIII) were measured by an enzyme-linked immunosorbent assay (ELISA). Adjustments including variables with different baseline distribution at univariate level with P<0.1 (age, body mass index, coronary artery disease, fibrinogen) were performed in multivariate models. Higher collagen XVIII was found in AAA patients in comparison with the control group of patients (39.5 vs. 25.0 ng/mL; P=0.002). Diameter of abdominal aorta was positively associated with collagen XVIII levels in univariate (B=0.16; P=0.004), and in multivariate analysis (B=0.14; P=0.027), i.e. increase in collagen XVIII by 1 ng/mL corresponded to an increase in abdominal aortic diameter by 0.14 mm. Patients with serum collagen XVIII levels in the third tertile (˃47 ng/mL) had 4.23 times higher risk of AAA compared to patients with collagen XVIII levels in the first and second tertiles (OR 4.23; 95% CI 1.42-11.6; P=0.020). No association was found between other examined biomarkers and abdominal aortic diameter. Diameter of abdominal aorta was positively associated with serum collagen XVIII level.

The potential use of Endostatin and Angiopoietin-2 as valuable biomarkers for the prediction of Diabetic Nephropathy in Type 2 Diabetes Mellitus

Diabetic Nephropathy (DN) is considered a serious capillary complication of diabetes mellitus (DM) which leads to end-stage renal disease. Endostatin (EST) is considered as collagen XVIII fragment formed during extracellular matrix remodeling (ECMR). EST acts as an anti-angiogenic factor. Angiopoietin-2 (Ang II) is a growth factor that increases in several conditions, such as hyperglycemia. The present study was to scrutinize the association of EST and Ang II serum levels with nephropathy in patients with type 2 diabetes mellitus (T2DM). A total of 30 healthy individuals (control) and 120 T2DM patients classified into 60 patients with microalbuminuria and 60 patients without microalbuminuria), aged 45-65 years were included. Fasting Plasma Glucose (FPG), HbA1C%, lipid profile, urinary albumin/creatinine ratio of 30-300 mg/g (UACR), serum urea and creatinine levels were assessed. Both EST and Ang II were measured using the ELISA technique. Ang II and EST levels were elevated in patients with T2DM groups compared with the healthy control group (P<0.001). EST and Ang II were significantly correlated to UACR (r= 0.753, P<0.001) (r= 0.685, P<0.001) and therefore indicate progress to DN. Circulating EST and Ang II were significantly associated with T2DM and predict progression of DN and therefore can be used as biomarkers for the prediction of DN in such a group of patients.

Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice.

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.

Circulating endostatin as a risk factor for cardiovascular events in patients with stable coronary heart disease: A CLARICOR trial sub-study

Background and aimsRaised levels of serum endostatin, a biologically active fragment of collagen XVIII, have been observed in patients with ischemic heart disease but association with incident cardiovascular events in patients with stable coronary heart disease is uncertain. MethodsThe CLARICOR-trial is a randomized, placebo-controlled trial of stable coronary heart disease patients evaluating 14-day treatment with clarithromycin. The primary outcome was a composite of acute myocardial infarction, unstable angina pectoris, cerebrovascular disease or all-cause mortality. In the present sub-study using 10-year follow-up data, we investigated associations between serum endostatin at entry (randomization) and the composite outcome and its components during follow-up. The placebo group was used as discovery sample (1204 events, n = 1998) and the clarithromycin-treated group as replication sample (1220 events, n = 1979). ResultsIn Cox regression models adjusting for cardiovascular risk factors, glomerular filtration rate, and current pharmacological treatment, higher serum endostatin was associated with an increased risk of the composite outcome in the discovery sample (hazard ratio per standard deviation increase 1.11, 95% CI 1.03–1.19, p = 0.004), but slightly weaker and not statistically significant in the replication sample (hazard ratio 1.06, 95% CI 1.00–1.14, p = 0.06). In contrast, strong and consistent associations were found between endostatin and cardiovascular and all-cause mortality in all multivariable models and sub-samples. Addition of endostatin to a model with established cardiovascular risk factors provided no substantial improvement of risk prediction (<1%). ConclusionsRaised levels of serum endostatin might be associated with cardiovascular events in patients with stable coronary heart disease. The clinical utility of endostatin measurements remains to be established.

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model.

Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

Estimation of Endostatin level in pulmonary arterial hypertension patients and its relation with some parameters

Abstract Biomarkers are attractive non-invasive tools for estimating and monitoring pulmonary arterial hypertension (PAH) disease and for predicting survival in patients with PAH; therefore, many studies encouraged the investigation of new biomarkers to facilitate the diagnosis of PAH. Endostatin (ES) is an endogenous inhibitor of angiogenesis. It is produced by proteolytic cleavage of the collagen XVIII that is present in both normal and cancerous tissue. In vitro examination shows that ES can manage endothelial cells (EC) physiology in ways that could influence angiogenesis. For example, solvent ES hinders EC movement and prompts improvements of the cytoskeleton that incorporate the loss of Actin stretch strands and central grips. This effect embraces restrictions on the α5β1integrins, Tropomyosin, and putative heparan sulfate proteoglycans. Consequences for the human EC cytoskeleton include Es-induced down-regulation of Mitogen-actuated Protein Kinase (MAPK), Focal Adhesion Kinase (FAK), the Urokinase Plasminogen Activator (uPA) System, and the RhoA GTPase. Human ES has likewise been shown in a few investigations to repress EC multiplication. Moreover, ES-instigated cell cycle capture in the G1 stage is joined by Cyclin D1 down-regulation. Of note, ES blocks the proliferation and organization of endothelial cells into new blood vessels, and in animal studies, ES also inhibits angiogenesis and the growth of both primary tumors and secondary metastasis. ES was initially identified by its capacity to inhibit tumor angiogenesis in vitro and also in vivo. It can also be found in both healthy and patient’ serum, and has been detected in peripheral circulation. ES could be an attractive, non-invasive prognostic marker for some diseases, notably PAH. Therefore, the presented work is aimed at investigating the ES level in blood serum as a biomarker for detection, diagnosis and early treatment of PAH patients. In doing so, the association is ascertained between gender, age, body mass index (BMI), waist circumferences, smoking, types of PAH (primary and secondary) and this potential biomarker is assessed in PAH patients.

The N-terminal domain of unknown function (DUF959) in collagen XVIII is intrinsically disordered and highly O-glycosylated.

Collagen XVIII (ColXVIII) is a non-fibrillar collagen and proteoglycan that exists in three isoforms: short, medium and long. The medium and long isoforms contain a unique N-terminal domain of unknown function, DUF959, and our sequence-based secondary structure predictions indicated that DUF959 could be an intrinsically disordered domain. Recombinant DUF959 produced in mammalian cells consisted of ∼50% glycans and had a molecular mass of 63 kDa. Circular dichroism spectroscopy confirmed the disordered character of DUF959, and static light scattering indicated a monomeric state for glycosylated DUF959 in solution. Small-angle X-ray scattering showed DUF959 to be a highly extended, flexible molecule with a maximum dimension of ∼23 nm. Glycosidase treatment demonstrated considerable amounts of O-glycosylation, and expression of DUF959 in HEK293 SimpleCells capable of synthesizing only truncated O-glycans confirmed the presence of N-acetylgalactosamine-type O-glycans. The DUF959 sequence is characterized by numerous Ser and Thr residues, and this accounts for the finding that half of the recombinant protein consists of glycans. Thus, the medium and long ColXVIII isoforms contain at their extreme N-terminus a disordered, elongated and highly O-glycosylated mucin-like domain that is not found in other collagens, and we suggest naming it the Mucin-like domain in ColXVIII (MUCL-C18). As intrinsically disordered regions and their post-translational modifications are often involved in protein interactions, our findings may point towards a role of the flexible mucin-like domain of ColXVIII as an interaction hub affecting cell signaling. Moreover, the MUCL-C18 may also serve as a lubricant at cell-extracellular matrix interfaces.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Rh-endostatin Concomitant with Chemotherapy Versus Single Agent Chemotherapy for Treating Soft Tissue and Bone Sarcomas: A Systematic Review and Meta-Analysis.

Endostar (recombinant human endostatin (rh-endostatin)), a 20-kDa proteolytic fragment of collagen XVIII, was approved for the treatment of non-small cell lung cancer (NSCLC). Recently, several studies have evaluated the efficacy of rh-endostatin combined with chemotherapy in the treatment of bone and soft tissue sarcomas. Here, we conducted a systematic review and meta-analysis to assess available evidence.Methods:Pubmed, Embase, Web of Sciences, the Cochrane Library and two Chinese literature databases (CNKI, WanFang) were systematically searched till May 20, 2018. Randomized controlled trials (RCTs) and cohort studies which compared the outcomes of rh-endostatin combined with chemotherapy versus chemotherapy alone for treating bone sarcomas or soft tissue sarcomas were included. The primary outcome was overall survival rate (OSR). Secondary outcomes included objectiveremissionrate(ORR), clinical benefit rate (CBR), disease control rate (DCR), distant metastasis rate (DMR) and adverse effects (AEs). The methodological quality of the included studies was evaluated. Data analysis was performed by Revman 5.3 software.Results:9 studies comprising 839 patients were included. The pooled results indicated that, compared with chemotherapeutic agents alone, rh-endostatin combined group had a significant benefit in 1-year and 2-year OSR. However, there were no difference between 5-year OSR. OR, CBR and DMR were higher in rh-endostatin combined group. No significant difference was observed in the incidence of AEs.Conclusions:Rh-endostatin combined chemotherapeutic agents significantly improved clinical efficacy compared with chemotherapeutic agents alone in treating bone and soft tissue sarcomas. Moreover, combination of rh-endostatin with chemotherapy didn't increase the incidence of AEs. But more high quality RCTs with large sample size should be done in the future to confirm the conclusion.

Ephrin-B3 binds both cell-associated and secreted proteoglycans

The ephrin family of membrane proteins binds Eph tyrosine kinase receptors. We have previously shown that ephrin-B3 also binds to heparan sulfate proteoglycans (HSPGs). We now show that ephrin-B3 can bind both secretory and cell associated PGs, such as agrin, collagen XVIII, Perlecan, and CD44, and indicate that such interaction with cell associated PGs involves a complex including 20 and 45 kDa proteins. Ephrin-B3 binding to HEK-293T cells is blocked by a secretory variant of CD44 (v3-v10), while over-expression of membrane associated CD44 increased ephrin-B3 binding. In addition, ephrin-B3 precipitated CD44 expressed by the oral squamous carcinoma cell line H376. Moreover, ephrin-B3 binding affinities to heparin and CD44 in solution was strong.In conclusion, we have identified secretory and cell associated PGs with high ability to bind ephrin-B3 and suggest that ephrin-B3 can bind to a protein complex organized by a membrane associated PG.

Serum Endostatin Levels in Oral Squamous Cell Carcinoma.

Endostatin is a C-terminal proteolytic fragment of collagen XVIII and, as with angiostatin and thrombospondin, is known as an antiangiogenic agent. The aim of this study was to assess the level of serum endostatin in patients with oral squamous cell carcinoma (SCC), and its association with the clinicopathological characteristics of the tumor. Using an enzyme-linked immunosorbent assay (ELISA) kit, we investigated the circulating levels of endostatin in the blood serum of 45 patients with oral SCC and 45 healthy controls. The mean level of serum endostatin in patients was significantly lower (68.8±85 ng/ml) than in healthy controls (175.6±73 ng/ml) (P<0.001). Serum endostatin levels were significantly lower in patients with lymph node metastasis compared with patients without lymph node metastasis (P<0.001). In addition, serum endostatin level was associated with higher histological grade (P<0.001). There were no apparent correlations between serum endostatin concentration and clinicopathological features such as age, gender, and tumor stage (P>0.05). Findings of the present study suggest the prognostic and anti-metastatic role of endostatin, and this may be used as a tool for monitoring tumor progression.

Collagen XVIII and LOXL-4 polymorphisms in women with and without advanced pelvic organ prolapse.

We verified the presence of single nucleotide polymorphisms (SNP) rs2236479 of the collagen 18 (COL18A1) and rs2862296 of the lysyl oxidase-like 4 (LOXL-4) genes and the association with pelvic organ prolapse (POP) in Brazilian women and determined risk factors for POP development. We assessed 532 postmenopausal women divided into POP (stages III and IV) and control (stages 0 and I) groups by examination and peripheral blood sample collection. DNA sequences of interest were analyzed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We used logistic regression models for the analyses, with p < 0.005 for significance. The frequency of homozygous polymorphic alleles (AA) in COL18A1 and (GG) in LOXL-4 were similar in both groups (17.5% and 15.4% for COL18A1 and 18.9% and 20.6% for LOXL-4, respectively). There were no associations between those polymorphisms or other genotypes and POP. Multiple logistic regression analysis identified age [odds ratio (OR) = 1.10, confidence interval (CI) 95% = 1.07; 1.14), number of vaginal births (OR = 1.66, CI 95% = 1.36; 2.03), and family history (OR = 2.55 CI 95% = 1.43; 4.55) as independent risk factors for POP. Our study suggests lack of association between DNA polymorphisms rs2236479 of COL18A1 and rs2862296 of LOXL-4 with advanced POP in this population.

The association between circulating endostatin and a disturbed circadian blood pressure pattern in patients with type 2 diabetes

Background: Endostatin, cleaved from collagen XVIII in the extracellular matrix, is a promising circulating biomarker for cardiovascular damage. It possesses anti-angiogenic and anti-fibrotic functions and has even been suggested to be involved in blood pressure regulation. Less is known if endostatin levels relate to circadian blood pressure patterns. In the present paper we studied the association between circulating levels of endostatin and nocturnal dipping in blood pressure.Methods: We used the CARDIPP-study, a cohort of middle aged, type 2 diabetics (n = 593, 32% women), with data on both 24-hour and office blood pressure, serum-endostatin, cardiovascular risk factors, and incident major cardiovascular events. Nocturnal dipping was defined as a >10% difference between day- and night-time blood pressures.Results: Two-hundred four participants (34%) were classified as non-dippers. The mean endostatin levels were significantly higher in non-dippers compared to dippers (mean ± standard deviation: 62.6 ± 1.8 µg/l vs. 58.7 ± 1.6 µg/l, respectively, p = .007). Higher serum levels of endostatin were associated with a diminished decline in nocturnal blood pressure adjusted for age, sex, HbA1c, mean systolic day blood pressure, hypertension treatment, glomerular filtration rate, and prevalent cardiovascular disease (regression coefficient per SD increase of endostatin −0.01, 95% CI, −0.02–(−0.001), p = .03). Structural equation modelling analyses suggest that endostatin mediates 7% of the association between non-dipping and major cardiovascular events.Conclusion: We found an independent association between higher circulating levels of endostatin and a reduced difference between day- and night-time systolic blood pressure in patients with type 2 diabetes. Yet endostatin mediated only a small portion of the association between non-dipping and cardiovascular events arguing against a clinical utility of our findings.

Collagen 18 and agrin are secreted by neural crest cells to remodel their microenvironment and regulate their migration during enteric nervous system development.

The enteric nervous system (ENS) arises from neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the intestinal wall. Many extracellular matrix (ECM) components are present in the embryonic gut, but their role in regulating ENS development is largely unknown. Here, we identify heparan sulfate proteoglycan proteins, including collagen XVIII (Col18) and agrin, as important regulators of enteric neural crest-derived cell (ENCDC) development. In developing avian hindgut, Col18 is expressed at the ENCDC wavefront, while agrin expression occurs later. Both proteins are normally present around enteric ganglia, but are absent in aganglionic gut. Using chick-mouse intestinal chimeras and enteric neurospheres, we show that vagal- and sacral-derived ENCDCs from both species secrete Col18 and agrin. Whereas glia express Col18 and agrin, enteric neurons only express the latter. Functional studies demonstrate that Col18 is permissive whereas agrin is strongly inhibitory to ENCDC migration, consistent with the timing of their expression during ENS development. We conclude that ENCDCs govern their own migration by actively remodeling their microenvironment through secretion of ECM proteins.

Comparison of the proteomes of mouse Skin Derived Precursors (SKPs) and SKP‐derived fibroblasts (SFBs) by iTRAQ

Skin-derived precursors (SKPs) can differentiate into fibroblasts. However, little is known about the molecular mechanism of the differentiation process. Our previous study has compared the transcriptomes of mouse SKPs and SKP-derived fibroblasts (SFBs) by RNA-Seq analysis and found some genes and signal pathways that might play important roles in the transition from SKPs to SFBs. Here, to further investigate the process by which SKPs differentiate into fibroblasts, we compared the proteomes between SKPs and SFBs using iTRAQ. Our results showed that 243 differentially expressed proteins (DEPs) were identified. Among the 28 DEPs which were related to the functional group of cell differentiation, Down-regulated DEPs collagen XVIII, keratin 10, keratin 5, keratin 14, and TIFIbeta, up-regulated DEPs Thy-1, ANXA1, alpha-catenin, and calreticulin might play important roles in the transition of SKPs to fibroblasts. Further study is needed to clarify the roles of these proteins in the differentiation process of SKPs into fibroblasts, which could facilitate investigations of the detailed molecular mechanisms in the process and make it possible to take advantage of the potential therapeutic applications of SKPs in skin regeneration in the future.

Polymicrogyria and Intractable Epilepsy in Siblings With Knobloch Syndrome and Homozygous Mutation of COL18A1

Knobloch and Layer described a triad of symptoms – vitreoretinal degeneration, high myopia, and congenital encephalocele – in 5 affected siblings in a large consanguineous family.1 The cause of what is now Knobloch syndrome (KS) was identified as mutation of COL18A1, the gene encoding collagen XVIII.2 We present an unusual case of two brothers with severe global developmental delay, medically intractable epilepsy, diffuse polymicrogyria, and high myopia, born to non-consanguineous parents of Palestinian ethnicity. Although the brothers lack typically-associated skull defects, they were found to harbor a novel homozygous pathogenic mutation in COL18A1, leading to a diagnosis of KS on the severe end of the phenotypic spectrum. Epilepsy and brain malformations are uncommon in cases of KS; hence, this report expands the current clinical spectrum and reinforces the diagnostic utility of trio whole exome sequencing analysis in appropriately selected cases of polymicrogyria.

Are Pro8/Pro18 really critical for functional dynamic behavior of human endostatin N-terminal peptide? A comparative molecular dynamics study

Endostatin which is derived from the non-collagenous domain 1 of collagen XVIII and is a recently identified broad spectrum anti-angiogenesis agent, inhibits 65 different tumor types. The N-terminal fragment of endostatin protein (ES) has the same antitumor, antimigration and antipermeability effects as the entire protein. In the current study, we modeled two mutant variants of ES with two mutation sites (M1-ES (Pro8 → Ala) and M2-ES (Pro18 → Ala)) and tried to understand proline’s effect on the peptide structure/stability by introducing P8A/P18A mutations, and then in order to gain functional insight into mutation caused by amino acid substitution to the peptide structure/function, these effects were predicted using computational tools. From the RMSD analyses, it can be concluded that dynamic behavior of wild-type and mutant structures was not significantly different from each other and all systems reached equilibrium. The RMSF analysis also revealed that the M2-ES has smaller overall flexibility than the WT-ES and M1-ES structures. The radius of gyration analysis then confirmed the structure of M2-ES compared to wild-type and M1 variant becomes more compact during simulation of our systems. Finally, molecular dynamics simulation analysis shows that replacement of Pro residue with Ala is able to induce a distinct β-sheet in both mutant structures. Indeed, the docking analysis shows the WT-ES and M2-ES bind to the same region of αvβ3 integrin, suggesting similar interaction pattern with a relatively equal binding energy into this receptor. Our results speculated that the P8A/P18A replacements confer no improvement (or no tangible weakness) in the peptide biological activity although is able to change structural conformation of N-terminal fragment of human endostatin protein.