Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: (A) Representative Movat Pentachrome-stained histologic images of the aortic and mitral valves of control and Prox1 ΔVEC mice at various ages. Glycosaminoglycans, collagen and elastin are stained in blue, red and black, respectively. (B) Quantification of aortic valve and mitral valve thickness. For each of the mice four to seven sections at 100-120 μm intervals were analyzed. The area of the valve leaflets (2 leaflets from base to tip) was measured for every section and their average was used as a measure of valve thickness. Data is represented as Mean ± SEM. Statistical significance was assessed using a two-tailed, unpaired t test. Each dot represents an individual mouse, n=8-23 mice/genotype/time point. **** = p < 0.0001; *** = p < 0.001; ** = p < 0.01; * = p < 0.05. (C) Echocardiography of 12-month-old control and Prox1 ΔVEC mice. Left: representative echocardiography images show decreased cusp separation (double-headed arrows in the top panels) and increased aortic peak velocity (bottom panels) in Prox1 ΔVEC mice. Right: bar charts of cusp separation and aortic valve peak velocity. No obvious defects were observed in left ventricle functions in the Prox1 ΔVEC mice as indicated by normal ejection fraction and fractional shortening. Data is represented as Mean ± SD. Statistical significance was assessed using a two-tailed, unpaired t test. n=5 control and n=9 Prox1 ΔVEC mice. * = p < 0.05. Abbreviations: AV, aortic valve; MV, mitral valve; A, aorta; V, left ventricle.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: (A) Representative scanning electron microscope (SEM) images of the tri-cuspid aortic valve leaflets of control and Prox1 ΔVEC mice. SEM images show that Prox1 ΔVEC mice have thicker aortic valves, disrupted endothelial layer (arrows in the middle row) with infiltration of platelet-like cells (arrows in the bottom row). N=5 for the control and N=6 for Prox1 ΔVEC mice. (B) Representative Movat Pentachrome-stained histologic images demonstrated thrombus-like structure in the aortic valve of a Prox1 ΔVEC mouse (arrow). The graph shows the quantification of the percentage of mice with thrombi in the aortic valves. Valves were analyzed by either SEM or Movat Pentachrome-staining of sections. N=10 control mice (5 by SEM, 5 by staining) and n=16 Prox1 ΔVEC mice (6 by SEM and 10 by staining). (C) vWF expression was increased in the downstream side VECs of Prox1 ΔVEC mice (arrows). N=5 for control mice; n=7 for Prox1 ΔVEC mice. (D) FOXC2 + VECs (arrows) were reduced in Prox1 ΔVEC mice. N=4 for control mice; n=5 for Prox1 ΔVEC mice. Data is represented as Mean ± SEM. Statistical significance was assessed using the Fisher exact test in (B) and a two-tailed, unpaired t test in (C) and (D). * = p < 0.05; **** = p < 0.0001. Each dot represents the average of 3-5 sections from a mouse.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Microscopy, Control, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: (A) qRT-PCR for the expression of selected cytokines and growth factors in the valve tissue from control and Prox1 ΔVEC mice. The dotted line represents normalized expression in control valves. Each dot represents aortic and mitral valve tissues collected from five 3-month-old mice. Data is represented as Mean ± SD. (B) Representative RNAscope images for Pdgfb expression (red dots) in the aortic valves of 3-month-old control and Prox1 ΔVEC mice. The lower panels are enlarged images of the boxed areas in the upper panels. Quantification of the RNAscope results showed that Prox1 ΔVEC mice had higher Pdgfb expression. Each dot represents the average from 3 sections of a mouse valve. N=4 mice per genotype. (C) Representative Movat Pentachrome-stained images of aortic valves from 6-month-old control and Prox1-2A-Cre;R26 +/LSL-PDGFB mice. Thicker aortic valves were observed in Prox1-2A-Cre;R26 +/LSL-PDGFB mice when compared with control valves. N=6 control and n=9 Prox1-2A-Cre;R26 +/LSL-PDGFB mice. (D) Representative Movat Pentachrome stain images of the aortic valves of 6-month-old control and Tie2-Cre ;Pdgfrb +/D849V mice. Tie2-Cre ;Pdgfrb +/D849V mice had enlarged aortic valves when compared to control valves. N=5 for control and Tie2-Cre ;Pdgfrb +/D849V mice. The area of valve leaflets was measured from 4-6 stained sections per valve. The average of these areas is represented as the thickness of valve in that mouse. Statistical significance was assessed using a two-tailed, unpaired t test in (B, C, and D) and using a one sample t and Wilcoxon test in (A). * = p < 0.05. ** = p < 0.01.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Quantitative RT-PCR, Expressing, Control, RNAscope, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: Knock down of FOXC2 results in enlarged aortic valves, and over expression of FOXC2 rescues the aortic valve defects of Prox1 ΔVEC mice. (A) Representative images of Movat Pentachrome staining that was performed using aortic valves of 12-month-old control and Foxc2 ΔVEC mice with graph showing the semi-quantitative measurement of valve thickness. N=8 controls and n=8 Foxc2 ΔVEC mice. (B) Representative images of Resorcin-Fuchsin staining that was performed using the aortic valves of 12-month-old control and Foxc2 ΔVEC mice. Green arrowhead shows the normal expression of elastin on the upstream side of valves. The orange arrowheads show abnormal elastin expression within and in the downstream side of valves. The graph shows the distribution of normal and abnormal elastin expression in control and mutant mice. N=11 controls and n=7 Foxc2 ΔVEC mice. (C) Representative immunofluorescence images for versican expression in the aortic valves of 12-month-old control and Foxc2 ΔVEC mice, followed by semi-quantitative measurement. N=10 control and n=8 Foxc2 ΔVEC mice. (D) Representative RNAscope images for Pdgfb expression (red dots) in the aortic valves of 6-month-old control and Foxc2 ΔVEC mice (arrows indicate Pdgfb + VECs). Quantification of the RNAscope results showed that Foxc2 ΔVEC mice had higher Pdgfb expression. Each dot represents the average of 3 sections from a mouse valve. N=3 control and n=2 Foxc2 ΔVEC mice. (E-H) Over expression of FOXC2 in Prox1 ΔVEC mice ( Prox1 ΔVEC ;Foxc2 GOF ) ameliorates valve thickness (E), abnormal elastin distribution (F), increased proteoglycan expression (G) and Pdgfb expression (H). N=13 control, n=11 Prox1 ΔVEC and n=7 Prox1 ΔVEC ;Foxc2 ΔGOF mice in (E); N=13 control, n=11 Prox1 ΔVEC and n=9 Prox1 ΔVEC ;Foxc2 ΔGOF mice in (F); N=8 control, n=12 Prox1 ΔVEC and n=7 Prox1 ΔVEC ;Foxc2 ΔGOF mice in (G). N=3 for all three genotypes in (H). Mice were 6-month-old in (E-G) and 3-month-old in (H). Data is represented as Mean ± SEM. Statistical significance was assessed using a two-tailed, unpaired t test in (A, C, and D), one-way ANOVA and Tukey’s multiple comparisons test in (E, G, and H), and Fisher exact test in (B and F). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Each dot represents the average of 3-5 sections from a mouse.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Knockdown, Over Expression, Staining, Control, Expressing, Mutagenesis, Immunofluorescence, RNAscope, Two Tailed Test

Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: (A, B) Protein and RNA samples were obtained from the mitral valves of MVP patients (n=77) who underwent mitral valve replacement surgery. Genes and protein expressions were quantified by qRT-PCR and ELISA respectively. Pearson correlation method was used for calculating r and p. (C) Aortic valve leaflets from 3 patients with aortic valve insufficiency were histologically analyzed. The relatively normal-looking leaflet was used as the internal control. The normal and pathological leaflets were analyzed using Movat Pentachrome stain and by immunohistochemistry for PDGF-B, SOX9, and PROX1. (D-F) Expression of PDGF-B and SOX9 was increased in the diseased leaflet, but no significant difference was observed in the number of PROX1 + cells. In PROX1 panels white arrows indicate PROX1 + VECs and yellow arrows indicate PROX1 + cells of unknown identity in the valve interstitium. Data is represented as Mean ± SEM. * = p <0.05. Statistical significance was assessed using a two-tailed, unpaired t test. Quantification was performed by averaging the data from 5 sections. PDGF-B staining was quantified as a percentage of PDGFB + CD31 + cells to total CD31 + cells. Abbreviations: AVI, leaflet with aortic valve insufficiency.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Staining, Immunohistochemistry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

doi: 10.1101/2023.05.10.540284

Figure Lengend Snippet: (A) Schematic of the experimental design. Imatinib (50mg/kg-BW/day) or PBS (vehicle) was orally administrated to control and Prox1 ΔVEC mice daily for 24 weeks starting from when they were 1-month-old. At the end of the treatment aortic valves were evaluated by echocardiography and histology. (B) Echocardiography showed that imatinib enhanced cusp separation and lowered aortic peak velocity in Prox1 ΔVEC mice when compared to untreated Prox1 ΔVEC mice. There were no significant differences in left ventricle functions (ejection fraction and fractional shortening). N=7 vehicle treated controls, n= 8 vehicle treated Prox1 ΔVEC mice, n=5 imatinib treated controls and n=5 imatinib treated Prox1 ΔVEC mice. (C) Representative Movat Pentachrome-stained sections from the aortic valves of control and Prox1 ΔVEC mice treated with vehicle or imatinib. The graphs show the quantification of valve thickness and the presence of thrombi. Arrow points to a thrombus-like structure in an untreated Prox1 ΔVEC mouse. The number of thrombus-like structures was reduced in Prox1 ΔVEC mice treated with imatinib. The area of valve leaflets was measured from 4-6 sections per valve. The average of these areas is represented as the thickness of valve in that mouse. Each dot represents an individual mouse. Valve thickness in Prox1 ΔVEC mice was not reduced by imatinib treatment. N=12 vehicle treated controls, n=10 vehicle treated Prox1 ΔVEC mice, n=5 imatinib treated controls and n=5 imatinib treated Prox1 ΔVEC mice. (D) Abnormal elastin distribution was rescued in the aortic valves of imatinib treated Prox1 ΔVEC mice. N=10 vehicle treated control, n=10 vehicle treated Prox1 ΔVEC , n=5 imatinib treated control and n=5 imatinib treated Prox1 ΔVEC mice. (E) IHC and quantification indicated that abnormal versican production in Prox1 ΔVEC mice was inhibited by imatinib treated. N=6 vehicle treated controls, n=10 vehicle treated Prox1 ΔVEC mice, n=4 imatinib treated controls and n=5 imatinib treated Prox1 ΔVEC mice. Data is represented in Mean ± SD in (B) and Mean ± SEM in (C and E). Statistical significance was assessed using a two-way ANOVA and Tukey’s multiple comparisons test in (B, C, and E) and using the Fisher exact test in (C and D). * = p <0.05; ** = p <0.01, *** = p <0.001, **** = p <0.0001 and. Each dot represents the average of 3-5 sections from a mouse. Abbreviations: Veh, vehicle; Imb, imatinib.

Article Snippet: Movat pentachrome (Abcam; ab245884), Alizarin red (IHC World; IW-3001) and Resorcin-Fuchsin (Electron Microscopy Sciences; 26370-01) staining’s were performed using the manufacturer’s protocols.

Techniques: Control, Staining