Kindlin‐3 Promotes Angiogenesis via Notch Signalling and Is Crucial for Functional Recovery Postmyocardial Infarction

Figures

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  FIGURE 1. Kindlin‐3 expression increases in CMECs after MI. (A, B) Quantitative PCR (qPCR) analysis showing Kindlin‐3 mRNA expression in cardiac tissues and CMECs from the infarct border zone at different time points post‐MI (n = 6). (C) Representative images and quantitative analysis of Kindlin‐3 protein expression in CMECs from the infarct border area post‐MI (n = 3). (D) Representative immunohistochemical staining of Kindlin‐3 in Sham‐operated and myocardial infarction (MI) mice, including the remote area (RA) and border area (BA) post‐MI (Scale bars = 20 μm). Kindlin‐3 expression levels were quantified and compared (n = 4). (E) qPCR analysis of Kindlin‐3 mRNA expression in Sham‐operated and MI mice, including the remote area (RA) and border area (BA) post‐MI (n = 4). (F) Immunoblotting analysis showing representative images and quantitative data of Kindlin‐3 expression in CMECs under hypoxia or normoxia conditions (n = 6). (G) The effects of hypoxia on tube formation were assessed using a Matrigel assay (n = 3). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01 versus sham or normoxia group.

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  FIGURE 2. Kindlin‐3 treatment improves cardiac function and reduces cardiomyocyte apoptosis in the post‐MI border zone. Kindlin‐3 was administered via the tail vein as described in the Materials and Methods section. (A) The peak early filling to late diastolic ventricular filling velocity (E/A ratio) was used to assess cardiac diastolic function, (B) while ejection fraction (EF%) and fractional shortening (FS%) were used to evaluate cardiac systolic function (n = 6). (C) Representative images of TUNEL and nuclear (DAPI) staining in cardiomyocytes (α‐actinin) from AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA). Arrows indicate TUNEL‐positive (green) cardiomyocyte (red) nuclei (blue; Scale bars = 20 μm; n = 6). (D) Representative Western blot images and quantitative analysis showing the ratio of Cleaved Caspase 3 to Caspase 3 in mice 28 days post‐MI (n = 3). ns, not significant. Data were presented as mean ± SEM. **p < 0.01.

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  FIGURE 3. Kindlin‐3 promotes angiogenesis in vivo. (A) Representative images of Masson's trichrome staining and quantitative data illustrating the extent of fibrosis in heart sections from the indicated groups (n = 6). Scale bars = 500 μm. (B) Four weeks post‐MI, ventricular tissues were analysed using immunofluorescence staining to evaluate capillary (CD31) and arteriolar density (α‐SMA) in AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA, n = 6). (C) Representative images and quantitative analysis of Western blots demonstrate the expression of angiogenesis‐related proteins in mice 28 days post‐MI (n = 3). Data were presented as mean ± SEM. ns, not significant, *p < 0.05, **p＜0.01.

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  FIGURE 4. Kindlin‐3 promotes angiogenesis in vitro. (A) Cell viability in CMECs was analysed using the CCK‐8 assay at 0, 24 and 72 h following Kindlin‐3 overexpression or knockdown (n = 3). (B) Scratch assays and tube formation were assessed through representative images and quantitative analysis under conditions of Kindlin‐3 overexpression or knockdown (n = 3). (C) Representative Western blot images and quantitative analysis demonstrating Kindlin‐3 protein levels and angiogenesis‐related protein expression in CMECs following transfection with Kindlin‐3 overexpression plasmids (n = 3). (D) Representative Western blot images and quantitative analysis showing Kindlin‐3 protein levels and angiogenesis‐related protein expression in CMECs after transfection with siRNA targeting Kindlin‐3 (n = 3). Data were presented as mean ± SEM. **p < 0.01.

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  FIGURE 5. Kindlin‐3 overexpression activates the Notch signalling pathway. (A) Representative immunohistochemical staining of Notch1 in AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA). (Scale bars = 20 μm). Notch1 expression levels were quantified and compared (n = 6). (B) qPCR analysis of Notch1 mRNA expression in AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA) (n = 4). (C) Representative Western blot images and quantitative analysis showing Notch1, Hes1, and Hif‐1α protein levels in CMECs. ns, not significant. Data were presented as mean ± SEM. **p < 0.01, *p < 0.05.

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  FIGURE 6. Inhibition of the Notch pathway abolishes Kindlin‐3 enhanced angiogenesis. (A) DAPT treatment's impact on migration and tube formation was examined in Vector and K3‐OE CMECs (n = 3). (B) Representative images and quantitative analysis of Western blots demonstrate Notch1 and angiogenesis‐related protein levels in CMECs. DAPT 10 μM. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01.

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  FIGURE 7. Kindlin‐3 modulates Notch signalling through a β1 integrin‐dependent mechanism in vitro. (A) Representative immunohistochemical staining of β1 Integrin in AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA). (Scale bars = 20 μm). β1 Integrin expression levels were quantified and compared (n = 6). (B) qPCR analysis of β1 Integrin mRNA expression in AAV‐NC and AAV‐K3 mice post‐MI or Sham operation, including the remote area (RA) and border area (BA). (n = 4). (C) Quantitative analysis and representative Western blot images of β1‐Integrin in CMECs following treatment with Vector or K3‐OE (n = 3). (D) The expression of the specified proteins was evaluated through Western blotting after a 2‐h treatment with the Notch inhibitor DAPT. (n = 3). ns, not significant. Data were presented as mean ± SEM. **p < 0.01, *p < 0.05.