Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Abstract 16950: Exosomes Derived From Podoplanin Positive Cells Induce Fibrosis and Inflammation in Healthy Mouse Heart

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derived fromactivated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase. In vivo,PDPN-exo were initially injected into the left ventricle of healthy mouse hearts followed withexosomes boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase ofthe end diastolic and systolic volumes. The fibrotic area was characterized by vessels doublepositive to endothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Inconclusion these data suggest that PDPN-exo alter the biology of mCECs, fibroblast andmonocytes and participate in adverse remodeling after MI; their specific cargo may representa cohort of targets for the treatment of cardiac fibrosis.

Abstract 16837: Exosomes Derived From Podoplanin Positive Cells Induce Serum Amyloid A3 Amyloidosis in Healthy Mouse Heart

The extra-cellular-matrix (ECM) composition of scar tissue after myocardial infarction (MI)has been largely investigated; although fibronectin and collagen are favorable for newmyocyte formation there is a missing component that increase the stiffness and reducethe remodeling of the ischemic area. We identified a primary Serum Amyloid A3 (Saa3)extracellular accumulation that contribute to the chronic alteration of the tissue. Serumamyloid amyloidosis (AA) are characterized by deposition of hepatic misfolded protein,Saa3 is the only amyloid protein that is released locally after inflammation, mostly bymesenchymal progenitor cells. We already described that two days after MI,mesenchymal and endothelial progenitor cells express Podoplanin (PDPN) a plateletaggregation-inducing type I transmembrane glycoprotein as a signal of activation. Exosomes derived from this cohort of cells actively release Saa3 in the ECM affecting thebiology of fibrosis beyond the inflammation. Specific histological staining such asthioflavin t and Congo red, showed amyloid deposition in mouse hearts 1 month after MI;furthermore, immunohistochemistry for Saa3 detected the deposition of the misfoldedprotein alongside fibronectin and collagen. PDPN+ cells were isolated 2 days after MI,cultured expanded and activated with TNFα and AngiotensinII. Activated PDPN positivecells highly expressed Saa3 and exosomes derived from activated PDPN+ cellsconditioned media were used in vivo for the treatment of healthy mouse hearts. Treatedmice developed an extended epicardial fibrosis and amyloidosis with a subsequentimpairment in the contractility and increase of the end diastolic and systolic volumes. Thefibrotic area was characterized by infiltrating CD45+ cells. Novel therapies aimed atpromoting clearance of existing amyloid deposits may be an effective approach in thenear future in the treatment of scar remodeling after MI.

Abstract 467: Exosomes Derived From Podoplanin Positive Cells Alter Physiology and Structure of Healthy Mouse Heart

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derivedfrom activated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase andSerum Amyloid A3 protein accumulation/mis-folding. In vivo, PDPN-exo were initially injectedinto the left ventricle of healthy mouse hearts followed with exosomes boosters delivered byretro-orbital vein injection. Treated mice developed an extended epicardial fibrosis andamyloidosis with a subsequent impairment in the contractility and increase of the end diastolicand systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. In conclusionthese data suggest that PDPN-exo alter the biology of mCECs, fibroblast and monocytes andparticipate in adverse remodeling after MI; their specific cargo may represent a cohort oftargets for the treatment of cardiac fibrosis.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2–79.1%) and Janthinobacterium (0.7–13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

Introducing Murine Microbiome Database (MMDB): A Curated Database with Taxonomic Profiling of the Healthy Mouse Gastrointestinal Microbiome

The gut microbiota modulates overall metabolism, the immune system and brain development of the host. The majority of mammalian gut microbiota consists of bacteria. Among various model animals, the mouse has been most widely used in pre-clinical biological experiments. The significant compositional differences in taxonomic profiles among different mouse strains due to gastrointestinal locations, genotypes and vendors have been well documented. However, details of such variations are yet to be elucidated. This study compiled and analyzed 16S rRNA gene-based taxonomic profiles of 554 healthy mouse samples from 14 different projects to construct a comprehensive database of the microbiome of a healthy mouse gastrointestinal tract. The database, named Murine Microbiome Database, should provide researchers with useful taxonomic information and better biological insight about how each taxon, such as genus and species, is associated with locations in the gastrointestinal tract, genotypes and vendors. The database is freely accessible over the Internet at http://leb.snu.ac.kr/mmdb/.