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. 2023 Apr 3;11(1):67-71.
doi: 10.1016/j.gendis.2023.02.045. eCollection 2024 Jan.

Identification of galectin-3 as a novel potential prognostic/predictive biomarker and therapeutic target for cerebral cavernous malformation disease

Souvik Kar  1 , Andrea Perrelli  2   3   4 , Kiran Kumar Bali  5 , Raffaella Mastrocola  2   3 , Arpita Kar  6   7 , Bushra Khan  8 , Luis Gand  8 , Arnab Nayak  8 , Christian Hartmann  9 , Wolfram S Kunz  10 , Amir Samii  1 , Helmut Bertalanffy  1 , Saverio Francesco Retta  2   3
Affiliations

Identification of galectin-3 as a novel potential prognostic/predictive biomarker and therapeutic target for cerebral cavernous malformation disease

Souvik Kar et al. Genes Dis. .
No abstract available

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Figures

Fig. 1
Figure 1
Gal-3 expression levels were significantly up-regulated both in surgical CCM specimens and in blood samples of CCM patients. Moreover, they were inversely correlated with the expression levels of KRIT1 in both cellular and animal models. (A) Transcriptome profiles showing 4928 protein-coding genes (PCGs) significantly differentially expressed in human CCM tissues. The heatmap shows the differential expression of PCGs between human CCM specimens (n = 10) and control brain tissues (n = 4) (GEO accession number: GSE137596). Up-regulated and down-regulated expressions are indicated in red and green colors, respectively. Galectin-3 (LGALS3) was identified among the significantly up-regulated PCGs in human CCM tissues, as highlighted in the inset image. Statistical significance was calculated by a two-tailed Student's t-test, and a log-fold change cutoff of ±1.5 and a P value of < 0.01 were applied to select the most significantly differentially expressed PCGs. (B–E) A significant increase in Gal-3 mRNA expression levels occurred both in CCM lesions and blood samples of CCM patients and was reflected at the protein level. (B, C) Bar diagrams representing the fold change mean ± SEM in Gal-3 mRNA levels in human brain CCM tissue samples (CCM; n = 4) vs. paired controls (Control; n = 3) (B), and in blood samples of CCM patients (CCM; n = 5) vs. healthy controls (Control; n = 4) (C), as determined by qRT-PCR gene expression assays. The housekeeping genes GAPDH (B) and Rpl37a (C) were used as internal reference controls for normalizing the relative Gal-3 mRNA expression levels. ∗∗P < 0.001, Student's t-test of three independent assays. (D) Representative Western blot analysis of Gal-3 protein (∼30 kDa) expression levels in three distinct surgically resected human CCM lesions (CCM) and control brain tissues (Control). β-tubulin (∼52 kDa) was used as internal loading control for normalizing the relative Gal-3 protein expression levels. (E) Bar diagram representing the relative Gal-3 protein expression levels in human CCM lesions (CCM) vs. control brain tissues (Control). Semiquantitative densitometric analysis of protein bands on Western blots was performed with the ImageJ software, and relative quantification values were expressed as a ratio of the mean optical density (OD) of each Gal-3 band in CCM vs. control samples upon normalization with the OD of corresponding β-tubulin bands (n = 3 per group). Note that Gal-3 mRNA levels were significantly up-regulated both in CCM lesions (B) and blood samples (C) of CCM patients. Moreover, the significant increase in Gal-3 mRNA expression detected in CCM lesions (B) was clearly reflected at the protein level (D, E) (∗∗P < 0.0005). Statistical significance was calculated by a two-tailed Student's t-test, and a P-value ≤ 0.05 was considered statistically significant. (F) Immunohistochemical analysis showing that Gal-3 was highly expressed both in endothelial cells lining human CCM lesions and in infiltrating leukocytes. (F, panel a) Representative hematoxylin and eosin (H&E) staining of a histological section from a surgically resected human CCM lesion showing a cluster of abnormally enlarged capillaries (caverns) filled with blood cells and lined by a thin endothelium layer (arrows). (F, panel b) Negative immunohistochemical control, incubated with non-immune serum, showing no staining. (F, panel c) Representative immunohistochemical analysis of Gal-3 expression in histological sections obtained from surgically resected human CCM lesions. Note that there was positive Gal-3 staining in endothelial cells lining a large CCM cavern (solid arrowheads). Focal Gal-3 staining was also observed within the perilesional brain tissue (star). (F, panel d) Human CCM lesion showing infiltrating leukocytes as dark brown dots positive for Gal-3 antibody (arrows). (G, H) Gal-3 expression is up-regulated in the brain of KRIT1 heterozygous mice. (G) Representative Western blot analysis of KRIT1 and Gal-3 expression levels in brain hemispheres of 26-week-old KRIT1 heterozygous (KRIT1+/−) mice and wild-type (WT) littermates. The housekeeping α-tubulin protein was used as an internal reference control for normalizing the relative KRIT1 and Gal-3 protein expression levels. Two biological replicates are shown for each sample and are representative of three independent experiments. (H) Bar diagrams representing the densitometric analysis of Gal-3 and KRIT1 Western blot bands (left and right panels, respectively) upon normalization with the OD of corresponding α-tubulin bands (n = 6 per group). Semiquantitative densitometric analysis of Western blot bands was performed with the ImageJ software, and relative quantification values were expressed as a normalized ratio of Gal-3 and KRIT1 OD mean with respect to α-tubulin. Note that the down-regulation of KRIT1 protein levels in the brain of KRIT1+/− mice vs. WT littermates was associated with a significant upregulation of Gal-3 protein levels (∗∗P < 0.05). Statistical significance was calculated by a two-tailed Student's t-test, and a P-value ≤ 0.05 was considered statistically significant. (I–L) Gal-3 expression was strongly up-regulated in both KRIT1-silenced endothelial cells and KRIT1-knockout MEF cells. (I) Representative Western blot analysis of KRIT1 and Gal-3 expression levels in human umbilical vein endothelial cells (HUVECs) subjected to two rounds of transfection with short interfering RNAs (siRNAs) against KRIT1 (KRIT1 siRNA) or scrambled control siRNAs (Ctrl siRNA). The housekeeping β-tubulin protein was used as an internal reference control for normalizing the relative KRIT1 and Gal-3 protein expression levels. (J) Bar diagrams representing the densitometric analysis of Gal-3 and KRIT1 Western blot bands (left and right panels, respectively) upon normalization with the OD of corresponding β-tubulin bands (n = 6 per group). (K) Representative Western blot analysis of KRIT1 and Gal-3 expression levels in genetically homogeneous MEF cells either KRIT1-knockout (KRIT1-KO) or re-expressing KRIT1 at high levels (KRIT1+/+). The housekeeping α-tubulin protein was used as an internal reference control for normalizing the relative KRIT1 and Gal-3 protein expression levels. (L) Bar diagrams representing the densitometric analysis of Gal-3 and KRIT1 Western blot bands (left and right panels, respectively) upon normalization with the OD of corresponding α-tubulin bands (n = 6 per group). Two representative Western blot replicates are shown for both HUVEC (I) and MEF (K) cells, along with the densitometric quantification of all biological replicates (J, L). Semiquantitative densitometric analysis of Western blot bands was performed with the ImageJ software, and relative quantification values were expressed as a normalized ratio of Gal-3 and KRIT1 OD mean with respect to either β-tubulin or α-tubulin. Note that there was a highly significant inverse correlation between KRIT1 and Gal-3 protein expression levels (∗∗∗P < 0.0001). Statistical significance was calculated by a two-tailed Student's t-test, and a P-value ≤ 0.05 was considered statistically significant.
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References

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