Fig 1.
The functional network of proteins that differed significantly between CDC-treated and PBS-treated PAH animals.
Fig 2.
RV free wall vascularity assessment in PAH rats at 4 weeks post treatment.
(A) Representative images of capillaries (Isolectin B4, green) and arterioles (Isolectin B4 and smooth muscle actin, green and red) within the RV free wall from healthy, PBS-treated and CDC-treated PAH animals are shown. Examples of capillaries and arterioles are indicated by blue and white arrowheads, respectively. Analysis of the mean number of (B) arterioles and (C) capillaries per image (± SEM) are shown. Significant difference between the indicated groups (brackets) are represented by * (p<0.05), ** (p<0.01), ***(p<0.001). Note that some of the extracellular green signal observed in the PAH-PBS and PAH-CDC images may be from fibrosis autofluorescence. Scale bar = 50µm. (D) Angiogenesis-associated proteins that are differentially expressed in Healthy vs. PAH-PBS animals and PAH-CDC vs PAH-PBS animals are shown (cyan and orange bars, respectively), with bar direction indicating either up or down regulation. (E) Table listing the fold changes (log2(FC)) of proteins for the relevant samples. Darker purple colors (- numbers) represent downregulation and lighter pink colors (+ numbers) represent up-regulation of proteins. Values denoted by * represent proteins that were identified only in certain samples (only in CDC-treated or only in control sample), therefore may not be indicative of true changes.
Fig 3.
Histological and proteomic fibrosis assessment within the RV free wall at four weeks post treatment.
(A) Transverse cryosections of hearts from the three animal groups, stained with Picrosirius red to show the amount and locations of fibrosis, are shown. (B) RV free wall fibrosis (represented as a percentage of total RV tissue area) is shown for all animals, along with their mean values (horizontal bar) ± SEM. Significant difference between the indicated groups (brackets) are represented by * (p<0.05), ** (p<0.01), ***(p<0.001). (C) Fibrosis-associated, differential protein expression is represented by individual boxes with the cyan (PAH-PBS vs Healthy) and orange (PAH-CDC vs. PAH-PBS) boxes visually indicating up or down regulation across a central horizontal axis. (D) Table lists the fold changes (log2(FC)) of proteins for the relevant samples. Darker purple colors (- numbers) represent downregulation and lighter pink colors (+ numbers) represent up-regulation of proteins. Values denoted by * represent proteins that were identified only in certain samples (only in CDC-treated or only in control sample), therefore may not be indicative of true changes.
Fig 4.
Perivascular inflammation assessment in healthy, CDC- and PBS-treated PAH rat RV free wall.
Immunohistochemical analysis of RV free wall tissue sections from healthy, CDC-treated and PBS-treated PAH animals at 4 weeks post treatment are shown. (A) Panel of representative images taken from the three experimental groups for macrophage markers (top row: CD68+, second row: iNOS+, third row: CD163+) and T-cell markers (fourth row: CD4+, fifth row: CD8+). The small, white arrowheads indicate examples of the cells that were counted within each image). Graphical representations of the mean immune cell count per image (± SEM) are shown for (B) CD68+, (C) iNOS+, (D) CD163+, (E) CD4+, and (F) CD8+, for each of the three treatment groups. Significant differences between the indicated groups (brackets) are represented by * (p<0.05), ** (p<0.01), ***(p<0.001). Scale bar = 200µm. (G) Immune system-associated differential protein expression is represented by individual boxes with the cyan (PAH-PBS vs Healthy) and orange (PAH-CDC vs. PAH-PBS) bars visually indicating up or down regulation across a central horizontal axis. (H) Table lists the fold changes (log2(FC)) of proteins for the relevant samples. Darker purple colors (- numbers) represent downregulation and lighter pink colors (+ numbers) represent up-regulation of proteins. Values denoted by * represent proteins that were identified only in certain samples (only in CDC-treated or only in control sample), therefore may not be indicative of true changes.
Fig 5.
Cardiomyocyte hypertrophy assessment of the rodent RV free wall at 4 weeks post treatment.
(A) Representative images of cardiomyocyte cross-sections within the RV free wall of PBS- or CDC-treated PAH animals are shown. The cell membranes are shown in green, and the myofibrils are shown in red. Scale bar = 100um. (B) Analysis of the mean cross-sectional areas of cardiomyocytes (± SEM) from the different PAH treatment groups are shown. (C) Hypertrophy-associated protein expression is represented by individual boxes with the cyan (PAH-PBS vs. Healthy) and orange (PAH-CDC vs. PAH-PBS) bars visually indicating up or down regulation across a central horizontal axis. (D) Table lists the fold changes (log2(FC)) of proteins for the relevant samples. Darker purple colors (- numbers) represent downregulation and lighter pink colors (+ numbers) represent up-regulation of proteins. Values denoted by * represent proteins that were identified only in certain samples (only in CDC-treated or only in control sample), therefore may not be indicative of true changes.
Fig 6.
Association of the upstream protein regulator, MYC, with the RV DEPs between CDC and PBS-treated animals.
Fig 7.
Analysis of rodent body weight, RV chamber pressure, and RV function.
(A) Healthy and PAH rat body weight measurements from immediate post-hypoxia (baseline) until 4 weeks post CDC or PBS delivery. (B) RV systolic chamber pressure, measured at 4 weeks post therapeutic delivery, is shown in healthy, CDC-treated and PBS-treated animals. (C) Weekly echocardiography assessments of TAPSE for the three animal groups are shown. TAPSE measurements are displayed as the mean percent change from baseline ± SEM. One-way Anova analysis with multiple comparisons was conducted between the three rodent groups, at each timepoint. Significant difference between the indicated groups (brackets or lines) are represented by * (p<0.05), ** (p<0.01), and *** (p<0.001).