Mryanda Sopel

Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with Angiotensin II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C57Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to 7 days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD11b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD133). Bone marrow (BM) origin of infiltrating cells was assessed using GFP+ chimeric animals. Relative qRT–PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-β1, CTGF) as well as the chemokine stromal-derived factor (SDF)-1α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP+, myocardial-infiltrating cells were not GFP+. Transcript levels for SDF-1α were significantly elevated at 1 day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD133 staining of infiltrating cells and evident GFP+ cellular infiltration when exposing GFP+ BM chimeras to AngII. Furthermore, a significant number of CD133+/SMA+ cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.01). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.

Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis

Using an established model of myocardial hypertrophy and fibrosis after angiotensin II (AngII) infusion, our aim was to characterize the early cellular element involved in the development of myocardial fibrosis in detail. Male Lewis rats were infused with saline or AngII (0.7 mg/kg per day) for up to seven days. Collagen deposition and cellular infiltration were identified by histology stains. Infiltrating cells were grown in vitro and examined by flow cytometry and immunostaining. Chemokine expression was measured using qRT‐PCR. AngII infusion resulted in multifocal myocardial cellular infiltration (peak at three days) that preceded collagen deposition. Monocyte chemotactic protein (MCP)‐1 transcripts peaked after one day of AngII exposure. Using a triple‐labelling technique, the infiltrating cells were found to express markers of leucocyte (ED1+), mesenchymal [α‐smooth muscle actin (SMA)+] and haematopeotic progenitor cells (CD133+) suggesting a fibroblast progenitor phenotype. In vitro, ED1+/SMA+/CD133+ cells were isolated and grown from AngII‐exposed animals. Comparatively few cells were cultured from untreated control hearts, and they were found to be ED1−/SMA+/CD133−. We provide evidence that myocardial ECM deposition is preceded by infiltration into the myocardium by cells that express a combination of haematopoietic (ED1, CD133) and mesenchymal (SMA) cell markers, which is a characteristic of the phenotype of fibroblast precursor cells, termed fibrocytes. This suggests that fibrocytes rather than (as is often presumed) leucocytes may have effector functions in the initiation of myocardial fibrosis.

Treatment with Activated Protein C (aPC) Is Protective during the Development of Myocardial Fibrosis: An Angiotensin II Infusion Model in Mice

Aims Myocardial fibrosis contributes to the development of heart failure. Activated Protein C (aPC) is a circulating anticoagulant with anti-inflammatory and cytoprotective properties. Using a model of myocardial fibrosis second to Angiotensin II (AngII) infusion, we investigated the novel therapeutic function aPC in the development of fibrosis. Methods and Results C57Bl/6 and Tie2-EPCR mice were infused with AngII (2.0 µg/kg/min), AngII and aPC (0.4 µg/kg/min) or saline for 3d. Hearts were harvested and processed for analysis or used for cellular isolation. Basic histology and collagen deposition were assessed using histologic stains. Transcript levels of molecular mediators were analyzed by quantitative RT-PCR. Mice infused with AngII exhibited multifocal areas of myocardial cellular infiltration associated with significant collagen deposition compared to saline control animals (p<0.01). AngII-aPC infusion inhibited this cellular infiltration and the corresponding collagen deposition. AngII-aPC infusion also inhibited significant expression of the pro-fibrotic cytokines TGF-β1, CTGF and PDGF found in AngII only infused animals (p<0.05). aPC signals through its receptor, EPCR. Using Tie2-EPCR animals, where endothelial cells over-express EPCR and exhibit enhanced aPC-EPCR signaling, no significant reduction in cellular infiltration or fibrosis was evident with AngII infusion suggesting aPC-mediate protection is endothelial cell independent. Isolated infiltrating cells expressed significant EPCR transcripts suggesting a direct effect on infiltrating cells. Conclusions This data indicates that aPC treatment abrogates the fibrogenic response to AngII. aPC does not appear to confer protection by stimulating the endothelium but by acting directly on the infiltrating cells, potentially inhibiting migration or activation.

Myocardial migration by fibroblast progenitor cells is blood pressure dependent in a model of angII myocardial fibrosis.

Activation of the renin–angiotensin system (RAS) is thought to promote myocardial fibrosis. However, it is unclear whether this physiological fibrotic response results from chronic hemodynamic stress or from direct cellular signaling. Male C57B/6 mice were randomly assigned to receive angiotensin II (AngII) (2.0 μg kg−1 min−1), AngII+hydralazine (6.9 μg kg−1 min−1) or saline (control) via osmotic pumps for 7 days. Blood pressure was measured via noninvasive plethysmography. Hearts were harvested and processed for analysis. Cellular infiltration and collagen deposition were analyzed using histological staining. Molecular mediators were assessed using quantitative RT-PCR. As previously described, animals that received AngII developed hypertension and multifocal cellular infiltration by SMA+/CD133+ fibroblast progenitors followed by collagen deposition. The coadministration of hydralazine with AngII completely inhibited the hypertensive effects of AngII (P⩽0.01) and resulted in minimal cellular infiltration and minimal collagen deposition. These findings were in the context of persistent RAS activation, which was evidenced by elevation in serum aldosterone levels in animals that received AngII or AngII+hydralazine compared with animals that received saline. At the molecular level, infusion of AngII resulted in the significant upregulation of profibrotic factors (connective tissue growth factor-7.8±0.7 fold), proinflammatory mediators (TNFα-4.6±0.8 fold; IL-1β-6.4±2.6 fold) and chemokines (CCL2-3.8±1.0 fold; CXCL12-3.2±0.4 fold), which were inhibited when hydralazine was also infused. We provide evidence that myocardial infiltration by fibroblast progenitor cells secondary to AngII and the resultant fibrosis can be prevented by the addition of hydralazine. Furthermore, the beneficial effects of hydralazine were observed while maintaining RAS activation, suggesting that the mechanism of fibrosis is blood pressure dependent.

Highly purified human peripheral blood monocytes produce IL-6 but not TNFα in response to angiotensin II

Hypothesis: Monocytes produce pro-inflammatory cytokines in response to Angiotensin II (AngII). Introduction: AngII has been suggested by many to be pro-inflammatory and likely to contribute to the migration of leukocytes in patients with cardiovascular conditions. Materials and methods: Monocytes were purified from peripheral blood mononuclear cells (PBMCs) by negative selection using antibodies conjugated to magnetic beads. Detection of CD14+ and AT1R expression was achieved by double-labeling flow cytometry. Highly purified monocytes were then stimulated with AngII (6 and 24 h) to assess IL-6 and TNF-α transcript levels by qRT-PCR and protein secretion by ELISA. Results: Monocytes comprised 9.7 ± 2.0% of the PBMCs. Monocyte isolation by negative selection yielded a purity of up to 99.8%. We demonstrated AT1R expression on 9.5 ± 0.3% of highly purifed CD14+/CD16- monocytes. Stimulation of highly purified monocytes with AngII resulted in increased transcript levels of IL-6 at 6 h but not at 24 h, and increased secretion of IL-6 in a dose-dependent manner compared with controls (p <0.01). Conversely, there was no increase in TNF-α mRNA transcripts or protein secretion. Conclusions: We provide evidence that a CD14+/CD16- subset of highly purified human monocytes express AT1R and respond to AngII exposure in vitro by producing IL-6 but not TNF-α.

Integrins and monocyte migration to the ischemic myocardium.

ABSTRACT Aims: Characterize mononuclear cell migration after acute-myocardial infarction (MI). Material and Methods: Male Lewis rats underwent a left thoracotomy and ligation of the left anterior descending coronary artery (MI group). Control animals underwent thoracotomy without ligation (Sham group). Animals were sacrificed at 0, 2, 4, or 24 hr after the onset of ischemia. Leukocyte migration was assessed using isolated and In111 labeled mononuclear cells (injected at the onset of ischemia) and γ-count determined at 24 hours. Inhibition of migration was evaluated with monoclonal anti α4 and/or β2 antibodies. Results: Serum troponin was significantly elevated in animals with MI as compared with Sham (p = .017). Labeled mononuclear cell migration was five-fold higher in MI-treated animals than in Sham (p = .006). ED-1 positive mononuclear cells were confirmed in the left myocardium after 24 hr of ischemia. MCP-1 mRNA was significantly elevated in the left myocardium at 2 hr and 4 hr and peaked at 24 hr (p <.05). In addition, α4 integrin blockade inhibited labeled mononuclear cell migration by 22%. Blockade of β2 integrin inhibited mononuclear cell migration by 48%, while the combined α4+β2 blockade resulted in 59% inhibition of labeled mononuclear cell migration compared with treatment with isotype control antibody (p = .001). Conclusions: Significant ED1+ mononuclear cell migration within 24 hr of MI correlated with peak MCP-1 mRNA. Monoclonal antibody blockade suggested that early mononuclear cell migration is dependent only in part on α4 and β2 integrins.