Figure 1.
Electrocardiography of the heart at 20 minutes, 30 minutes, 60 minutes, 4-hour and 24-hour following MI.
Representative ECG of 20 minutes, 30 minutes, 60 minutes, 4 hours and 24 hours post MI groups showing ST elevation compared to normal ECG in sham operated groups.
Figure 2.
Left ventricular galectin-1 and HIF-1 α concentration and plasma concentration of galectin-1 following MI.
A. The graph represents HIF-1 α concentrations at 20 min, 30 min, 60 min, 4 hours and 24 hours post myocardial infarction with corresponding sham operated groups in C57BL6 mouse left ventricle. Control represents non-operated normal animal heart. *Shows p<0.05. B. HIF-1 α levels in nuclear and cytoplasmic extracts of LV mouse heart at 20 minutes following MI. *Shows p<0.05. C. The graph represents left ventricular galectin-1 concentrations at 20 min, 30 min, 60 min, 4 hours and 24 hours post myocardial infarction with corresponding sham operated groups in C57BL6 mouse heart. Control represents non-operated normal animal heart. *Shows p<0.05. D. The graph represents ratio of plasma galectin-1 concentration in myocardial infarction to plasma galectin-1 concentration in sham operated mice C57BL6 mice. 4 hours and 24 hours post myocardial infarction show significant difference from sham operated mice. *Shows p<0.05. E. Pattern of Galectin-1 and HIF-1 α in the heart from 20 min post MI till 24 hour post MI time points, showing similar pattern of expression during the first 24-hour following MI.
Table 1.
HIF-1 α levels in pg/mg of total protein at different time points post myocardial infarction.
Table 2.
HIF-1 α levels in pg/mg total protein in nuclear and cytoplasmic fractions of left ventricular heart tissue at 20 min post myocardial infarction.
Figure 3.
HIF-1 α expression of the heart.
A. Representative section of naïve heart showing nuclear expression of HIF-1 α by few endothelial cells (arrow head), streptavidin- biotin immunoperoxidase method. B. Negative control section showing no HIF-1 α staining, streptavidin- biotin immunoperoxidase method. C. Positive control section of mouse placenta showing nuclear staining of HIF-1 α by trophoblastic cells, Rhodamine, immunofluorescent technique. D. Negative control section for HIF-1 α. Rhodamine, immunofluorescent technique. E,I,M&Q shows representative sections from the anterior wall of left ventricle in the area supplied by LAD 20 min, 30 min, 60 min and 4 hours following ligation of LAD, showing variable nuclear staining of HIF-1 α by cardiac myocytes at different time points (arrow head) and endothelial cells (thin arrow), streptavidin- biotin immunoperoxidase method. F,J,N&R shows their corresponding Sham operated hearts showing low nuclear expression of HIF-1 α by few endothelial cells (thin arrow), streptavidin- biotin immunoperoxidase method. Low and high power views of the left ventricle 20 min (G&H), 30 min (K&L), 60 min (O&P) and 4 hours (S&T) following ligation of LAD showing high nuclear staining of HIF-1 α by cardiac myocytes (arrow head), Rhodamine, immunofluorescent technique.
Figure 4.
Galectin-1 and HIF-1 α expression 24 hours following ligation of LAD.
A. A high power view of left ventricle in an area supplied by LAD showing coagulative necrosis (thin arrows) accompanied by heavy neutrophil polymorphs infiltration (arrow heads), H&E. B. A low power view of the heart showing areas of high expression of galectin-1 (thick arrows) surrounding areas of no expression (star shape) in the left ventricle and interventricular septum in the area supplied by LAD, streptavidin- biotin immunoperoxidase method. C. Sham operated heart showing lower expression of galectin-1 in the left ventricle and right ventricle (thick arrows), streptavidin- biotin immunoperoxidase method. D. Negative control section of the heart showing absence of staining of galectin-1. E, F&G. Representative section of the left ventricle from area supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thick arrow), surrounding areas of no expression (thin arrow). Many neutrophil polymorphs are also showing cytoplasmic expression of Galectin-1(arrow head), Streptavidin- biotin immunoperoxidase method. H, I&J. Representative section from left ventricle showing a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thick arrow) surrounding areas of no expression (thin arrow), many neutrophil polymorphs (arrow head) show cytoplasmic expression of Galectin-1, Alexa Fluor 488 immunofluorescent technique. K. Representative section from left ventricle showing nuclear expression of HIF-1 α by cardiac myocytes (arrow head) and neutrophil polymorphs (thin arrow), Rhodamine, immunofluorescent technique. L. Representative section from left ventricle showing cytoplasmic expression of Galectin-1 by cardiac myocytes (thick arrow), endothelial cells (arrow head) and neutrophil polymorphs (thin arrows), Alexa Fluor 488 immunofluorescent technique. M. Co-localization of Galectin-1 and HIF-1 α in cardiac myocytes (thick arrow), endothelial cell (arrow head) and neutrophil polymorphs (thin arrow), Alexa Fluor-Rhodamine immunofluorescent technique.
Table 3.
Galectin-1 levels in ng/mg of total protein at different time points post myocardial infarction.
Figure 5.
Galectin-1 expression in naïve heart.
I, Naïve heart, A. Low power view of the heart showing the left ventricle (arrow head), interventricular septum (thick arrow) and right ventricle wall (thin arrow), H&E stain. B&C are representative sections of the left and right ventricles respectively, H&E stain. D. showing low and focal expression of galectin-1 in the wall of the right ventricle (thin arrow) and the interventricular septum (thick arrow). E. A representative section of left ventricle showing mild expression of galectin-1 by endothelial cells (arrow head). F. A representative section of right ventricle showing low cytoplasmic expression of galectin-1 by cardiac myocyte (arrow head) and endothelial cells (thin arrow). G, H&I, immunofluorescent labeling of sections of the heart showing a net-like pattern of galectin-1 staining of cardiac myocytes (thin arrow) and cytoplasmic staining of endothelial cells (arrow head).
Figure 6.
Galectin-1 expression 20 minutes following ligation of LAD.
A. A low power view of the heart showing a high expression of galectin-1 in the anterior wall of left ventricle in the area supplied by LAD (thin arrow) and the interventricular septum (thick arrow). There is also increase in the expression of galectin-1 in the right ventricle (arrow head), streptavidin- biotin immunoperoxidase method. B. Sham operated heart showing lower expression of galectin-1 in the left ventricle and right ventricle (thin arrow), streptavidin- biotin immunoperoxidase method. C. Negative control section of the heart showing absence of galectin-1 staining. D, E&F. Representative sections of the left ventricle from area supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow D). High power views show a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow) and endothelial cells (arrow head, E&F), There is also nuclear expression of galectin 1 by cardiac myocytes (thick arrow E&F),streptavidin- biotin immunoperoxidase method. G, H&I. Representative section from left ventricle shows a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow).There is also high expression of galectin-1 in the Z bands (arrow head), Alexa Fluor 488 immunofluorescent technique.
Figure 7.
Galectin-1 expression 30 minutes following ligation of LAD.
A. A low power view of the heart showing a high expression of galectin-1 in the anterior wall of left ventricle in the area supplied by LAD (thin arrow) and the interventricular septum (thick arrow). There is also increase in the expression of galectin-1 in the right ventricle (arrow head), streptavidin- biotin immunoperoxidase method. B. Sham operated heart showing lower expression of galectin-1 in the left ventricle and right ventricle (thin arrow), streptavidin- biotin immunoperoxidase method. C. Negative control section of the heart showing absence of staining of galectin-1. D. A galectin-1 positive control section from prostate gland showing cytoplasmic expression of galectin- 1 by prostatic acini. E, F & G. Representative sections of the left ventricle from areas supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow), streptavidin- biotin immunoperoxidase method. H, I&J. Representative sections from left ventricle showing a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow), Alexa Fluor 488 immunofluorescent technique.
Figure 8.
Galectin-1 expression 60 minutes following ligation of LAD.
A& B, A low power view of the heart showing a high expression of galectin-1 in the anterior wall of left ventricle in the area supplied by LAD (thin arrow) and the interventricular septum (thick arrow). There is also increase in the expression of galectin-1 in the right ventricle (arrow head), streptavidin- biotin immunoperoxidase method. C. Sham operated heart showing low expression of galectin-1 in the left ventricle and right ventricle (thin arrow), streptavidin- biotin immunoperoxidase method. D. Negative control section of the heart showing absence of staining of galectin-1. E, F & G, Representative sections of the left ventricle from areas supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow) surrounding areas of low or absence of expression (star shape), streptavidin- biotin immunoperoxidase method. H, I& J, Representative section from left ventricle showing a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow), Alexa Fluor 488 immunofluorescent technique.
Figure 9.
Galectin-1 expression 4 hours following ligation of LAD.
A.A low power view of the heart showing a high expression of galectin-1 in the anterior wall of left ventricle in the area supplied by LAD (thin arrow) and the interventricular septum (thick arrow). There is also increase in the expression of galectin-1 in the right ventricle (arrow head), streptavidin- biotin immunoperoxidase method. B. Sham operated heart showing low expression of galectin-1 in the left ventricle (thin arrow) and right ventricle (arrow head), streptavidin- biotin immunoperoxidase method. C. Negative control section of the heart showing absence of staining of galectin-1. D. Representative section of the left ventricle from area supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow) surrounding areas of low or no expression (star shape), streptavidin- biotin immunoperoxidase method. E&F, Representative section of the left ventricle from area supplied by LAD showing a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow), streptavidin- biotin immunoperoxidase method. G, H&I. Representative section from left ventricle showing a well demarcated area of high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow) surrounding areas of low or no expression (star shape), Alexa Fluor 488 immunofluorescent technique.
Figure 10.
Co-localization of Galectin-1 and HIF-1 α.
A,D,G,J,M, Q show representative sections of the left ventricle from areas supplied by LAD in 20 minutes(Low power view), 20 minutes(high power view), 30 minutes, 60 minutes, 4 hours and 30 minutes (High power view) following MI respectively, showing high cytoplasmic expression of galectin-1 by cardiac myocytes (thin arrow), Alexa Fluor 488 immunofluorescent technique. B,E,H,K,N,W show representative sections of the left ventricle from areas supplied by LAD in 20 minutes(Low power view), 20 minutes(high power view), 30 minutes, 60 minutes, 4 hours and 30 minutes (High power view) following MI respectively showing high nuclear expression of HIF-1α by cardiac myocytes (thin arrow), Rhodamine immunofluorescent technique. C,F,I,L,P,X shows Co-localization of galectin-1 and HIF-1 α in the same sections at the respective time points. (arrow head), Alexa Fluor-Rhodamine immunofluorescent technique.
Figure 11.
Co-localization of Galectin 1, HIF-1 α, CD31, desmin and CD68.
A,D,G, Representative section of the left ventricle from areas supplied by LAD showing high cytoplasmic expression of galectin-1 by cardiac myocytes, endothelial cells and histiocytes respectively (thin arrows). J, showing high cytoplasmic expression of CD68 in tissue histiocytes (thin arrows) and M showing high cytoplasmic expression of CD31 in endothelial cells (thin arrows). Alexa Fluor 488 immunofluorescent technique. B. showing high cytoplasmic expression of desmin by cardiac myocytes (thin arrow), E. showing high cytoplasmic expression of CD31 by endothelial cells (thin arrow), H. showing high cytoplasmic expression of CD68 by histiocytes (thin arrow), K. showing high nuclear expression of HIF-1 α by histiocytes (thin arrow), N. showing high nuclear expression of HIF 1 α by endothelial cells (thin arrow), Rhodamine, immunofluorescent technique. C. Co-localization of galectin-1 and desmin in cardiac myocytes. F. Co-localization of galectin-1 and CD31 in endothelial cells (arrow head), I. Co-localization of galectin-1 and CD68 in histiocytes (arrow head), L. Co-localization of CD68 and HIF-1 α in tissue histiocytes (arrow head), O. Co-localization of CD31 and HIF-1 α in endothelial cells (arrow head). Alexa Fluor 488-Rhodamine immunofluorescent technique.
Table 4.
Morphometric analysis of expression of GAL-1 and HIF-1α in cardiomyocytes, endothelial cells and neutrophil polymorphs at different time points following ligation of LAD.
Figure 12.
Ki-67 proliferative activity in the left ventricle.
A, B, C, D, Low Ki-67 proliferative activity in normal left ventricle showing nuclear staining of Ki-67 in one interstitial cell (arrow head). E, F, G, 20 minutes MI; I,J, K, 30 minutes MI; M,N,O 60 minutes MI; Q,R,S, 4-hour MI, Low Ki-67 proliferative activity: showing nuclear staining of Ki-67 in few endothelial cells (arrow head) in left ventricle. U, V, W, 24-hour MI, high Ki-67 proliferative activity showing nuclear staining of Ki-67 in a large number of endothelial cells (arrow head) in the infarction area of left ventricle. H, L, P, T, X, showing faint nuclear staining of Ki-67 in one endothelial cell in 20 minutes, 30 minutes, 60 minutes, 4-hours and 24 hour sham-operated left ventricle. A, E, I, M, Q, U are stained by Streptavidin-Biotin immunoperoxidase method. The others are stained by Alexa Fluor 488 immunofluorescent technique.
Figure 13.
Apoptotic activity in the left ventricle.
A. showing high expression of caspase- 3 in_cardiac myocytes (arrow head) surrounding the necrotic area, endothelial cells (thin arrow), and neutrophil polymorphs (thick arrow) in 24-hour post MI, Alexa Fluor 488 immunofluorescent technique. B. showing no expression of caspase 3 in 24-hour sham-operated left ventricle. C, showing high expression of cleaved caspase- 3 in cardiac myocytes (thick arrows). Many apoptotic bodies (arrow head) are seen in the infarcted area of left ventricle, streptavidin- biotin immunoperoxidase method. D, showing very low apoptotic activity, only one cell stained with anti-cleaved caspase 3 (arrow head) in the area of infraction in the left ventricle of 4-hour post MI, streptavidin- biotin immunoperoxidase method. E, F, Showing no staining with anti-cleaved caspase 3 in the left ventricle of 30 minutes and 60 minutes post MI, streptavidin- biotin immunoperoxidase method.
Figure 14.
Bcl2 activity in left ventricle.
A, B, C, D, showing high cytoplasmic expression of bcl2 by cardiac myocytes (thick arrow), and endothelial cells (arrow head) at 30 minutes post MI in an area supplied by LAD in the left ventricle, streptavidin- biotin immunoperoxidase method. E, F, showing very low expression of bcl2 in few endothelial cells (arrow head) in the left ventricle of naïve and 30-minutes sham operated heart. Streptavidin- biotin immunoperoxidase method.
Figure 15.
Potential HREs in the promoter region of galectin-1 gene.
A, Human GAL-1 promoter with consensus sequence ACGTG (-1075). B, Human GAL-1 promoter with consensus sequence CACGC (−519, −533, −935). C, Mouse GAL-1 promoter with consensus sequence NCGTG (−978). D, Mouse GAL-1 promoter with consensus sequence TGCAC (−640).