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Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60-100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically S-35-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.

Heparanase is elevated in various pathological conditions, primarily cancer and inflammation. To investigate the significance and involvement of heparanase in liver fibrosis, we compared the susceptibility of wild-type (WT) and heparanase-overexpressing transgenic (Hpa-tg) mice to carbon tetrachloride (CCL4)-induced fibrosis. In comparison with WT mice, Hpa-tg mice displayed a severe degree of tissue damage and fibrosis, including higher necrotic tendency and intensified expression of smooth muscle actin. While damage to the WT liver started to recover after the acute phase, damage to the Hpa-tg liver was persistent. Recovery was attributed, in part, to heparanase-stimulated autophagic activity in response to CCL4, leading to increased apoptosis and necrosis. The total number of stellate cells was significantly higher in the Hpa-tg than the WT liver, likely contributing to the increased amounts of lipid droplets and smooth muscle actin. Our results support the notion that heparanase enhances inflammatory responses, and hence may serve as a target for the treatment of liver damage and fibrosis. De två första författarna delar förstaförfattarskapet.

Additional file 1: Supplemental Figure 1. Confirmation of mouse and human Hpa encoding genes expressions across all experimental conditions. (a) Genotype identification of Hpa- Tg mice. Semiquantitative PCR amplification of DNA extracted from WT (n=1) and Hpa-Tg mice (n=10). Human Hpa specific primer was used to detect transgene vector sequences in the mouse genome. Total DNA was assessed by specific primer against a genomic sequence of the ribosomal protein L-19. The expression levels of mouse Hpa (b) and human Hpa (c) in WT+SW/WT+Cn/Hpa-Tg+SW/Hpa-Tg+Cn group were confirmed. The expression levels of mouse vs human Hpa in in Hpa-Tg SW/HpaTg+Cn groups were confirmed (d). (e) Before 16S rRNA sequencing of WT and HpaTg mice, the expression levels of mouse Hpa and human Hpa were confirmed. The expression levels of mouse or human Hpa, the expression levels of mouse vs human Hpa were also confirmed in ABX (f), FMT (g-i), cohousing (j), Parabacteroides administration (k-m) and acetate supplementation (n-p) experiments. Data were expressed as mean ± SEM. Significance between two groups was determined by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Significance between multiple groups was determined through ordinary one-way ANOVA or Kruskal-Wallis test depending on the sample distribution. Exact p levels were all provided. ABX, antibiotic cocktail; Cn, caerulein; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; P. distasonis, Parabacteroides distasonis; SiHo, housed singly; SW, sterile water; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 2. Alpha-diversity, beta-diversity and bar plots of the phylum/class/order/family/genus taxonomic levels in WT and Hpa-Tg mice. (a) Alpha-diversity (Based on ACE, Observed features, Fisher alpha, Shannon and Faith pd). (b) PCoA of beta-diversity using Jaccard, Generalized Unifrac and Weighted Unifrac metric distance. (c) Quantification of dissimilarity values based on (b), presented as dissimilarity values (first (box bottom), third (box top) quartiles, the median (line inside box) and 2.5 interquartile range (line ends)). (d) Bar plots of the phylum taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. The relative abundances of Firmicutes and Bacteroidetes were shown. (e) Bar plots of the class taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. (f) Bar plots of the order taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 0.01% were analyzed. (g) Bar plots of the family taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 0.1% were analyzed. (h) Bar plots of the genus taxonomic levels in WT and Hpa-Tg mice. Relative abundance is plotted for each group. Bacterial genera with relative abundance greater than 1% were analyzed. Data were expressed as mean ± SEM. n=15 individuals/group. Significance between two groups was determined by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. For (b-c), differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities Hpa-Tg, heparanase-transgenic; WT, wild-type. Supplemental Figure 3. Exacerbated acute pancreatitis in Hpa-Tg mice depended on gut microbiota. (a) ABX experimental design, WT and Hpa-Tg littermates were put on a course of intragastrically antibiotic cocktail administration for 5 days for gut microbiota depletion After that, 16S rRNA sequencing analysis and AP induction were performed. (b) Pancreas (g)/body (g) weight x 1‰. (c) Serum amylase. (d) Serum lipase. (e) Representative images of pancreatic H&E staining. (f) Histologic score. (g) Serum TNF-α measured by ELISA. (h) Serum IL-6 measured by ELISA. (a-h) n=5 individuals/group. Data were expressed as mean ± SEM. Differences of data were assessed by assessed by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Exact p levels were all provided. Scale bars, 500 and 200 μm, respectively. ABX, antibiotic cocktail; AP, acute pancreatitis; Cn, caerulein; Hpa-Tg, heparanase-transgenic; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 4. Beta-diversity among ABX experimental groups. 16S rRNA sequencing analysis in fecal bacterial DNA from ABX WT mice (n=5) and Hpa Tg mice (n=3) was performed. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ABX, antibiotic cocktail; ANOSIM, analysis of similarities; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 5. Beta-diversity among FMT experimental groups. 16S rRNA sequencing analysis in fecal bacterial DNA from FMT groups was performed. n=7 individuals/group. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis FMT, fecal microbiota transplantation; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 6. Beta-diversity among CoHo WT and CoHo Hpa-Tg groups. 16S rRNA sequencing analysis in fecal bacterial DNA from CoHo WT and CoHo Hpa-Tg groups was performed. n=7 individuals/group. PCoA of beta-diversity using Bray-Curtis (a), Jaccard (b), Generalized Unifrac (c) and Weighted Unifrac (d) metric distance. Differences of data were assessed by ANOSIM test. Exact p levels were all provided. ANOSIM, analysis of similarities; CoHo, cohoused; Hpa-Tg, heparanase-transgenic; PCoA, principal coordinate analysis; rRNA, ribosomal RNA; WT, wild-type. Supplemental Figure 7. Parabacteroides was the most important biomarker to distinguish WT and Hpa-Tg mice. (a) VIP score of OPLS-DA. VIP score (calculated based on 16S rRNA sequencing data of Fig. 2) was used to rank the ability of different taxa to discriminate between WT and Hpa-Tg mice. A taxon with VIP score >1.5 was considered important in the discrimination. n=15 individuals/group. (b) In ABX experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing (n=5/3 in ABX WT/ABX Hpa-Tg group, respectively) and qRT-PCR (using specific primers of P. distasonis, n=5 individuals/group). (c) In FMT experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing and qRT-PCR (using specific primers of P. distasonis). n=7 individuals/group. (d) In Cohousing experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing and qRT-PCR (using specific primers of P. distasonis). n=7 individuals/group. (e-j) The correlations between severity indicators of AP and Parabacteroides abundance were analyzed using Spearman’s correlations. 6 mice each in WT and Hpa-Tg mice were included in the statistics. (k) In Parabacteroides administration experiment, Parabacteroides abundance was confirmed by 16S rRNA sequencing. n=5 individuals/group. Data were expressed as mean ± SEM. Differences of data in two groups were assessed by assessed by unpaired, two-tailed t test or Mann-Whitney test depending on the sample distribution. Differences of data in more than two groups were assessed by ordinary one-way ANOVA or Kruskal-Wallis test depending on the sample distribution. Exact p levels were all provided. ABX, antibiotic cocktail; AP, acute pancreatitis; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; OPLS-DA, orthogonal partial least squares discrimination analysis; P. distasonis, Parabacteroides. distasonis; qRT-PCR, Quantitative RT-PCR; rRNA, ribosomal RNA; VIP, valuable influence on projection; WT, wild-type. Supplemental Figure 8. Acetate concentration in ABX, FMT and cohousing experiments. (a) The whole annotation of microbial gene function of WT and Hpa-Tg mice on KEGG pathway analysis. Analysis of 16S rRNA sequencing data from Figure 2. n = 15 individuals/group. (b) Relative fold change of acetate concentration from cecal content in ABX experiment. n=5 individuals/group. (c) Relative fold change of acetate concentration from cecal content in FMT experiment. n=7 individuals/group. (d) Relative fold change of acetate concentration from cecal content in CoHo WT and CoHo Hpa-Tg mice. n=7 individuals/group. Data were expressed as mean ± SEM. Differences of data in two groups were assessed by assessed by Mann-Whitney test. Differences of data in FMT experiment was assessed by ordinary one-way ANOVA. Exact p levels were all provided. ABX, antibiotic cocktail; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; rRNA, ribosomal RNA; KEGG, kyoto encyclopedia of genes and genomes; WT, wild-type. Supplemental Figure 9. Confirmation of acetate enrichment in acetate supplementation experiment. Relative fold change of acetate concentration from cecal content in acetate supplementation experiment. n=9 individuals/group. Data were expressed as mean ± SEM. Differences of data were assessed by ordinary one-way ANOVA. Exact p levels were all provided. WT, wild-type; Hpa-Tg, heparanase transgenic. Supplemental Figure 10. Neutrophil infiltration depended on gut microbiota in heparanase-exacerbated acute pancreatitis. WT and Hpa-Tg mice were treated with/without Cn. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas were shown in (a) and (b), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of ABX experiment were shown in (c) and (d), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of FMT experiment were shown in (e) and (f), respectively. Representative plots of immunohistochemistry and bar plots of neutrophils in pancreas of cohousing experiment were shown in (g) and (h), respectively. Differences of data in two groups were assessed by unpaired, two-tailed t test. Differences of data in more than two groups were assessed by ordinary one-way ANOVA. Exact p levels were all provided. Scale bars, 200 and 50 μm, respectively. ABX, antibiotic cocktail; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase- X transgenic; Cn, caerulein; CoHo, cohoused. SiHo, housed singly; SW, sterile water; WT, wild-type. Supplemental Figure 11. Alpha rarefaction curves of all 16S rRNA sequencing analyses. Alpha rarefaction curves (Faith pd) in WT and Hpa-Tg groups (a), ABX experiment (b), CoHo WT and CoHo Hpa-Tg groups (c), FMT experiment (d) and the administration of Parabacteroides experiment (e). ABX, antibiotic cocktail; CoHo, cohoused; FMT, fecal microbiota transplantation; Hpa-Tg, heparanase-transgenic; P. distasonis, Parabacteroides distasonis; WT, wild-type. Supplemental Table 1. Schmidt's score system of pancreatic histopathology. Supplemental Table 2. Primers for qRT-PCR detection (SYBR Green). Supplemental Table 3. Primers for qRT-PCR detection (Taqman).

Additional file 1. Supplementary Table 1: Primary antibodies used in this study. Supplementary Table 2: The middle temporalis gyrus of Alzheimer’s disease and Non-demented control obtained from the Netherlands Brain Bank (NBB). Supplementary Table 3: The CSF and plasma samples of Alzheimer´s disease patients and non-demented controls from the Netherlands Brain Bank (NBB).