Evidence That Anemia Accelerates AS Progression Via Shear-Induced TGF-β1 Activation

Figures

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  Figure 1. AS Patients Have Higher Plasma Levels of Total TGF-Β1 Than Patient Control Subjects and TGF-Β1 Levels Correlate With Age in AS Patients (A) Total transforming growth factor (TGF)-β1 levels in plasma samples from healthy control subjects, patient control subjects, and aortic stenosis (AS) patients were measured by enzyme-linked immunosorbent assay. Individual subjects are shown as filled circles and plotted on a log scale with the median (Q1, Q3) shown as box and whisker plots. (B) Plasma TGF-β1 levels were positively correlated with age in AS patients (r = 0.47; P = 0.005), but not in patient control subjects (r = 0.12; P = 0.43). The P value for the group and age interaction in the analysis of variance model was 0.030, indicating a significant difference in the slope of the regression lines. (C) Total TGF-β1 levels in plasma of AS patients (n = 16) and patient control subjects older than 75 years of age (n = 11), and AS patients (n = 17) and patient-control subjects (n = 35) younger than 75 years of age. The older AS patients had higher levels than the older patient control subjects (P = 0.012) and the younger AS patients (P = 0.042). (D) Hemoglobin (Hb) levels in AS patients…

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  Figure 2. LA100 Mice, Which Spontaneously Develop AS, Have Higher Plasma Total TGF-β1 and Lower Hb Levels Than Control Mice (A) Plasma samples from control WT-C57Bl/6 and ApoE−/− mice (both do not develop AS; 25-125 weeks of age) and age-matched LA100 mice were assessed for total TGF-β1 levels by enzyme-linked immunosorbent assay. (B) Total TGF-β1 levels in plasma of older (>60 weeks) and younger (<60 weeks old) LA100 and control mice. Older LA100 mice had higher TGF-β1 than younger LA100 mice or older control mice (P < 0.0001) (C) Hb levels in older and younger LA100 and control mice. Older LA100 mice have lower Hb levels than younger mice (P < 0.0001) (D) Correlation between TGF-β1 and Hb levels in older LA100 mice (r = −0.417; P = 0.027). (E, F) AS progression parameters fractional valve openings and wall shear stress (WSS) measured by echocardiography in control and LA100 mice <60 and >60 weeks of age. Abbreviations as in Figure 1.

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  Figure 3. Anemia in LA100 Mice Is Associated With Higher Plasma Levels of Total and Active TGF-Β1 and AS Progression (A) Schematic diagram of anemia induction using low-iron diet and high-volume (300-500 μL) phlebotomy in <60 weeks) and >60 weeks of age LA100 mice. Control LA100 mice underwent low-volume (100 μL) phlebotomy. Weeks on diet and number of phlebotomies are indicated by blue bars. (B) Control LA100 mice <60 weeks of age were bled at low volume (100 μL) at baseline (B) and after 11 weeks. Hb levels in blood, collected at baseline (B) and at 11 weeks, were measured by HEMAVET HV950. Plasma total and active TGF-β1 levels were measured by enzyme-linked immunosorbent assay. (C) LA100 mice older and younger than 60 weeks were fed a low-iron diet followed by repeated high-volume (300-500 μL) phlebotomy up to 11 weeks as shown in A. Measurement of Hb levels at the indicated phlebotomy numbers showed progressive anemia with increasing phlebotomy numbers in both age groups (top). Plasma total and active TGF-β1 levels were measured by enzyme-linked immunosorbent assay, and both total and active TGF-β1 levels increase with the number of phlebotomies and correlated strongly with decreased…

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  Figure 4. Anemia Increases Plasma Total TGF-Β1 in WT-C57Bl/6 Mice, But Not in Mice With Targeted Inactivation of Tgfb1 in Platelets (PF4CreTgfb1flox/flox) (A) Schematic diagram of the phlebotomy protocol for WT-C57Bl/6 and PF4CreTgfb1flox/flox mice at 12 to 16 weeks of age fed a low-iron diet with large-volume (300-500 μL) phlebotomies are indicated by blue bars. (B) Assessment of Hb with subsequent phlebotomy demonstrated WT-C57Bl/6 and PF4CreTgfb1flox/flox mice became anemic compared with their baseline levels (P < 0.001). (C) Plasma total TGF-β1 levels at baseline and at 11 weeks after successive phlebotomies were measured by enzyme-linked immunosorbent assay. (D) Schematic diagram of the low-iron diet-induced mild anemia protocol for LA100 and LA100;PF4CreTgfb1flox/flox mice at 30 to 35 weeks of age fed a low-iron diet, and were bled low volume (100 μL) at baseline and after 8 weeks. (E) LA100 (30 weeks) and LA100;PF4CreTgfb1flox/flox (35 weeks) mice were fed with low-iron diet for 8 weeks. Measurement of Hb levels after 8 weeks showed mild anemia. (F) Plasma total TGF-β1 levels were measured by enzyme-linked immunosorbent assay and TGF-β1 levels increased in LA100 mice (P = 0.0016),…

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  Figure 5. LA100 Mice Develop Anemia and AVWS (A) Representative nonreduced immunoblot of plasma von Willebrand factor (vWf) analyzed in 1.5% agarose gel showing gradual reduction of vWf multimers with increasing numbers of phlebotomies in an LA100 mice. (B) Quantification of high molecular weight (HMW)-vWf multimers in LA100 mice showing gradual decrease in HMW-vWf multimers as anemia progressed (P < 0.0001). (C, D) HMW-vWf multimers correlated negatively with total (r = −0.540; P = 0.003) and active TGF-β1 (r = −0.496; P = 0.007) levels in anemic LA100 mice. (E) Whole-mount human AS-patient valve stained for RBCs (Ter119-green) and valvular cells nuclei (DAPI-blue) Arrows in high-magnification whole-mount staining and scanning electron microscopy images of aortic valve show RBC inside the damaged valve surface associated with platelet aggregates. (F) Whole-mount of LA100 mouse aortic valve leaflets stained for RBCs (Ter119-green) and for platelets (PF4-red) and valvular cells nuclei (DAPI-blue). Arrows in high-magnification whole-mount staining and scanning electron microscopy images show RBC inside the damaged valve surface associated with platelet aggregates.

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  Figure 6. LA100;C33S+/− and LA100;PF4CrePDIflox/flox Mice Are Partially Protected From AS Progression (A) Active TGF-β1 levels in serum from control LA100 and LA100;C33S+/− mice were measured by enzyme-linked immunosorbent assay after being subjected to shear rate of 1,800 s−1 for 2 hours. (B) Representative ultrasound images of aortic valve cusp (valve leaflets are marked with arrows) of LA100 and LA100;C33S+/− mice. (C) Fractional valve opening and wall shear stress (WSS) were measured by echocardiography in control, LA100 and LA100;C33S+/− mice >60 weeks of age. (D) Immunoblotting of platelet lysate from washed platelets with antibodies to protein disulfide isomerase (PDI) and actin showing PDI expression in platelets from WT and PF4CrePDIflox/flox mice. (E) Representative ultrasound images of aortic valve cusps of control, LA100 and LA100;PF4CrePDIflox/flox mice. (F) Fractional valve opening and WSS were measured by echocardiography in control LA100 and LA100;PF4CrePDIflox/flox mice >60 weeks of age.

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  Figure 7. Heyde’s Syndrome Heyde’s syndrome has traditionally been depicted as the linear sequence depicted in A. Our new data indicate that anemia itself can feed back to increase active TGF-β1 levels in AS patients and LA100 mice, as depicted in B, which in turn, can increase AS severity in a vicious circle. Abbreviations as in Figure 1.