Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity

Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood macrophage disappearance reaction. Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions

Changes in Dietary Fat Content Rapidly Alters the Mouse Plasma Coagulation Profile without Affecting Relative Transcript Levels of Coagulation Factors

<div><p>Background</p><p>Obesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events.</p><p>Objective</p><p>Establish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters.</p><p>Methods</p><p>Male C57BL/6J mice were fed a low fat (10% kcal as fat; LFD) or high fat diet (45% kcal as fat; HFD) for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F) VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding.</p><p>Results</p><p>HFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation.</p><p>Conclusions</p><p>Changes in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby preceding plasma metabolic changes, which cannot be explained by changes in relative expression of coagulation factors or decreased clearance rates.</p></div

Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.

<p>Data are expressed as mean (minimum-maximum expression level).</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD mice.</p><p>Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.</p

Regulation of the <i>F11, Klkb1, Cyp4v3</i> Gene Cluster in Livers of Metabolically Challenged Mice

<div><p>Single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that harbors the coagulation factor XI (<i>F11</i>), prekallikrein (<i>KLKB1</i>), and a cytochrome P450 family member (<i>CYP4V2</i>) genes are associated with deep venous thrombosis (DVT). These SNPs exert their effect on DVT by modifying the circulating levels of FXI. However, SNPs associated with DVT were not necessarily all in <i>F11</i>, but also in <i>KLKB1</i> and <i>CYP4V2</i>. Here, we searched for evidence for common regulatory elements within the 4q35.2 locus, outside the <i>F11</i> gene, that might control FXI plasma levels and/or DVT risk. To this end, we investigated the regulation of the orthologous mouse gene cluster under several metabolic conditions that impact mouse hepatic <i>F11</i> transcription. In livers of mice in which HNF4α, a key transcription factor controlling <i>F11</i>, was ablated, or reduced by siRNA, a strong decrease in hepatic <i>F11</i> transcript levels was observed that correlated with <i>Cyp4v3</i> (mouse orthologue of <i>CYP4V2</i>), but not by <i>Klkb1</i> levels. Estrogens induced hepatic <i>F11</i> and <i>Cyp4v3</i>, but not <i>Klkb1</i> transcript levels, whereas thyroid hormone strongly induced hepatic <i>F11</i> transcript levels, and reduced <i>Cyp4v3</i>, leaving <i>Klkb1</i> levels unaffected. Mice fed a high-fat diet also had elevated <i>F11</i> transcription, markedly paralleled by an induction of <i>Klkb1 and Cyp4v3</i> expression. We conclude that within the mouse <i>F11</i>, <i>Klkb1</i>, <i>Cyp4v3</i> gene cluster, <i>F11</i> and <i>Cyp4v3</i> frequently display striking parallel transcriptional responses suggesting the presence of shared regulatory elements. </p> </div

Effects low fat diet (LFD) and high fat diet (HFD) on plasma coagulation parameters.

<p>Effects on plasma coagulation parameters after 2 (panel A) and 16 (panel B) weeks of low fat diet (white) or high fat diet (black) feeding. Panel C shows the plasma coagulation profile of genetically obese <i>ob/ob</i> mice (striped) and their wild-type littermates (white) after 4 weeks on a low fat diet. Data are presented as mean±SEM. *p<0.05 and ^‡p<0.001 as compared to the LFD-fed mice or wild-type controls as appropriate.</p

Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.

<p>Data are expressed as mean (minimum-maximum expression level).</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD mice.</p><p>Hepatic mRNA levels of coagulation genes of mice on a low fat diet (LFD) or high fat diet (HFD) for 2 weeks.</p

Metabolic parameters of mice on a low fat diet (LFD) or high fat diet (HFD) for 16 weeks as compared to genetically obese <i>ob/ob</i> mice with their littermate wild-type controls after 4 weeks of LFD feeding.

<p>Data are expressed as mean±SEM.</p><p>*p<0.05 and</p><p>^‡p<0.001 as compared to LFD-fed mice or wild-type controls as appropriate.</p><p>Metabolic parameters of mice on a low fat diet (LFD) or high fat diet (HFD) for 16 weeks as compared to genetically obese <i>ob/ob</i> mice with their littermate wild-type controls after 4 weeks of LFD feeding.</p