Porphyrin‐Based SOD Mimic MnTnBuOE‐2‐PyP ⁵⁺ Inhibits Mechanisms of Aortic Valve Remodeling in Human and Murine Models of Aortic Valve Sclerosis

Figures

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  Figure 1. A, Alizarin red staining shows increased calcification in human sclerotic and stenotic aortic valves (n=5 per group). B, Bar graph shows fold‐change gene expression of CTGF (connective tissue growth factor), Cyr61 (cysteine‐rich angiogenic inducer 61), SPARC (secreted protein acidic and cysteine rich (osteonectin)), TSP (thrombospondin) ‐1 and ‐2, OPN (osteopontin) transcripts by reverse transcriptase–quantitative polymerase chain reaction (RT‐qPCR). Data were normalized against 18S (actin B) gene expression and represented as fold change+SE. *P, 0.01 (n=5 per group). C and D, Tissue preparation and representation of tensile uniaxial bioreactor, respectively. E, Immunofluorescence staining showing expression of Cyr61 and CTGF in sclerotic aortic valve tissue cultured in static condition in the presence of OS (oscillatory shear stress) + BMP4 (bone morphogenetic protein 4) (100 ng/mL; n=3 per group). F, RT‐PCR of OPN, Cyr61, and CTGF from RNA extracts collected from aortic valve cusps exposed to biomechanical stimulation (15% stretch at 1 Hz) for 6 days (n=5 per group). Osteogenic media enriched with BMP4 (100 ng/mL) was used.

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  Figure 2. A, Hematoxylin and eosin (H&E) staining of human aortic valves (AVs) explanted from controls and from AV sclerotic and stenotic patients. B, Picrosirius red staining of control, sclerotic, and stenotic leaflets was visualized using brightfield microscopy and polarized light (n=5 per group). Collagen accumulation was detected in spongiosa and ventricularis areas in both sclerotic and stenotic valves. C, Collagen fiber alignment maps at control, sclerotic, and stenotic stages. Yellow and red represent highly aligned tissue (n=5 per group) (D). E, Scanning electron microscopy was used to show alteration in the microstructure of diseased AV leaflets (n=3 per group). NOI indicates normalized orientation index.

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  Figure 3. A, Immunohistochemical staining of control, sclerotic, and stenotic leaflets shows distribution of nitrotyrosine (NO2Tyr) throughout leaflet, with areas of localized high‐intensity staining in aortic valve (AV) sclerotic and stenotic tissues (n=5 per group). B, Oxidized amino acid (OxAA) quantification in explanted leaflets measured by liquid chromatography–tandem mass spectrometry and expressed as μm/mol. C, Dityrosine (diTyr) quantification and calcium scores in control and AV sclerotic and stenotic leaflets. We conducted a Mann–Whitney U test to compare diTyr/tyrosine (diTyr/Tyr) in stenotic samples and controls (or sclerotic samples) and obtained a P value of 0.0075. BrTyr/Tyr indicates bromotyrosine/tyrosine; ClTyr/Tyr, Chlorotyrosine/tyrosine; diTyr/Tyr, dityrosine/tyrosine; m‐Tyr/Phe, meta‐tyrosine/phenylalanine; NO2Tyr/Tyr, nitrotyrosine/tyrosine; o‐Tyr/Phe, ortho‐tyrosine/phenylalanine.

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  Figure 4. A, Western blot showing the expression of H2AX, RUNX2, and SMA (smooth muscle actin) in whole‐cell extract of aortic valve (AV) sclerosis–derived valve interstitial cells treated or not with 500 μmol/L of H2O2. B, Bar graph shows fold‐change gene expression of α‐SMA evaluated by reverse transcriptase–quantitative polymerase chain reaction. Data were normalized against actin B and represented as fold change+SE. *Depicts statistically significant difference between Saline and Ang II (P<0.01). **Depicts statistically significant difference between Ang II+MnBuOE and Ang II (P<0.01). C, Relative AV thickness quantified using ImageJ with or without STDEV (n=5 per group) were used to evaluate differences in the thickness. D, Hematoxylin and eosin (H&E) staining of controls and AngII (angiotensin II) infused mouse AV tissues with or without MnBuOE or with MnBuOE alone. E, Collagen accumulation using picrosirius red staining. F, Picrosirius red staining of saline, Ang II, saline plus MnBuOE, and Ang II plus MnBuOE treated mice. G, Scanning electron microscopy used to show alteration in the microstructure of murine aortic valve leaflets.