The first description of MMP-like activity was related to the metamorphosis of the tadpole (Brinckerhoff and Matrisian, 2002). This and many other subsequent discoveries strongly suggest that this well-characterized family of metalloproteinases is also intimately involved in normal tissue growth and ECM remodeling (Stamenkovic, 2003). Morphogenesis and tissue growth, remodeling, and repair are sentinel features of childhood and adolescence.
Heart morphogenesis involves the characteristic mechanisms of cell proliferation, cell rearrangements, cell to cell, cell to matrix interactions, and tissue remodeling. Linask et al.
documented MMP activity regulates the coordination of early heart organogenesis by
affecting ventral closure of the heart and gut tubes, asymmetric cell proliferation in the dorsal mesocardium to drive looping direction, and ECM degradation within the dorsal mesocardium allowing looping to proceed toward completion (Linask et al., 2005), although little is known about the production, secretion, and clearance of these important proteinases throughout normal growth and development in human heart
At present, information suggests that the septum has both mesenchymal and muscular components (Contreras-Ramos et al., 2008). The mesenchymal element originates mainly from fusion of the conotruncal and atrioventricular endocardial cushions. Mechanisms that initiate development of the muscular septum are less well defined, but at least two processes have been proposed. The first process is that the muscular septum forms from coalescence of the part of the ventricular wall is interposed between the enlarging free walls of the
developing right and left ventricles; therefore, as the ventricular cavities become deeper the septum grows passively inwards ( Goor et al., 1970). The second process is an alternative hypo thesis suggesting that the muscular septum originates from a cluster of cells and the
so-called primitive inter ventricular septum, which expands actively towards the cushions of the atrioventricular canal (De La Cruz and Moreno-Rodriguez, 1998).
Gene polymorphism also is considered an essential cause of VSD formation; especially the TBX5 and GATA4 SNP are frequently discussed. These gene both express in the heart and their interaction is vital for normal cardiac septation (Maitra et al.,2009). TBX5 is a vital gene during embryonic differentiation which affects cardiac and upper limbs development.
When TBX5 occurs mutation may cause the loss of this gene function of and therefore cause cardiac and upper limbs malformations (Basson et al.,1997). A TBX5 polymorphismis also associated with ventricular septal defect in the Chinese Han population (Liu et al., 2009).
There are many researches point out that GATA4 gene mutationis related to the happen of ASD and VSD (Zhang et al., 2009; Chen et al., 2010b; Rajagopal et al.,2007). The other VSD related genes were list in Table 8. According to above, this research is based on studying the association of MMPs SNP and MMPs activity to the formation of VSDs and may participate the mechanism of spontaneous closure of VSDs.
To realize MMPs polymorphism from National Center for Biotechnology Information (NCBI)-SNP database, which had registered over 700 SNPs of MMP-2 and 300 SNPs of MMP-9. The relatively important and correlative literatures were organized and shown in Appendix 1 for MMP-2 and Appendix 2 for MMP-9. As MMPs play important role in many physiological functions, MMP polymorphism also has proven to be relative to many diseases.
Gene transcription is the primary point of regulation of MMPs, which implicates that sequence changes in the promoter may be important for the transcription and cause effect in protein levels and cell physiology.
Two of MMPs polymorphisms in promoter region were detected in this study: MMP-2 -735C>T and MMP-9 -1562 C>T. The reason of chosen these two promoter will described as
following. The C>T polymorphism, located at nucleotide -735, destroys a Sp1-binding element and the T allele is associated with significantly diminished promoter activity (Yu et al., 2004). The -1562 C>T polymorphism in the promoter region of MMP-9 exerts an T allelic effect on MMP-9 promoter strength and results in losing a nuclear repressor protein binding site which decreasing MMP- 9 expression as the T allele is present, and thus increasing the enzyme expression compared to the C allele (Morgan et al., 2003; Zhang et al., 1999).
Besides, if SNPs occurred at gene exon sequence, the structure of enzyme may change which further affects itself activity. So, this study also examined SNP in MMP-9 rs17576 (Q279R) and rs2250889 (R574P). SNP in MMP-9 rs17576 (Q279R) is a non-conservative amino acid substitution that modifies an amino acid residue within the highly conserved gelatinase-specific fibronectin type II domain (FN2) (Allan et al., 1995). Although the specific functional consequences of the substitution of proline for arginine coded by
rs2250889 in MMP-9 (R574P) have not been studied yet, the location of SNP rs2250889 in the C-terminal hemopexin-like domain could lead to an important protein structural
modification. This structural modification could either decrease the affinity of MMP-9 for its specific inhibitors, resulting in a higher level of protein activity, or enhance the affinity binding for gelatin which might increase its gelatinase activity (Rodríguez-Pla et al.,2008).
All SNPs above have more potential in ruling and causing MMP-2 and MMP-9 to significant change than other SNPs, which making them to be the target SNP in this research.
In this study, the SNPs of the MMPs genes, such as MMP-2 (-735C>T) and MMP-9 (-1562C>T, R279Q, and R574P) were determined by the polymerase chain reaction, restriction fragment length polymorphism method (Table 2). The results showed that genotype distribution and allele frequencies of MMP-2 -735C>T between the VSD and the control group had no significant differences (Table 3). This result suggest that C>T
polymorphism located at nucleotide -735 may not influence the incidence of VSD or not the core factor in Taiwan individuals.
MMP-2 activity in VSD patient plasma were also examined by zymography, categorized by their different genotypes (CC, CT, and TT) of MMP-2 -735 polymorphism and conducted in statistic. As mentioned earlier that C>T polymorphism located at nucleotide -735 should significantly diminish promoter activity (Yu et al., 2004); however, the statistical results in this study showed that there was no significant correlation between MMP-2 -735 genotypes and their activity. The result also showed that the T allele of MMP-2 -735 polymorphism has the highest MMP-2 activity which is different from the results of Yu C et al, suggesting C allele enhanced MMP-2 protein transcription which corresponds with higher MMP-2 activity (Yu et al., 2004).
Polymorphisms in the promoter of MMP-9 have been implicated in the regulation of gene expression and susceptibility to various diseases. The results of genotype distribution and allele frequencies of MMP-9 -1562 C>T between the VSD and the control group were shown in Table 4. The results show that polymorphism in the promoter of MMP-9 -1562 C>T has no correlation to VSD. However, there is no VSD patient have TT genotype of MMP-9 -1562 C>T in our study. We speculate that lack of T allelic genotype of MMP-9
polymorphism is relative to population and region, which is correspond to other disease studies conducted in Asian population also had few or even no T allelic genotype of MMP-9 polymorphism in their samples (Chen et al., 2010a; Lacchini et al., 2010; Buss et al., 2009).
The polymorphism at position -1562 were expected to change the promoter activity of MMP-9 (Zhang et al., 1999; Van den Steen et al., 2002); however, our results showed no relationship between genotypes of MMP-9 -1562 polymorphism and their activity. Our results
communicates with Demacq et al. whose plasma samples were recruited from healthy subjects (Demacq et al., 2006).
In detection of MMP-9 codon polymorphisms: MMP-9 Q279R and R574P, our data demonstrated that there is an association of the MMP-9 Q279R polymorphism and risk of VSD (Table 5 and Table 6). The importance of MMP-9 Q279R is based on the ability of changing MMP-9 protein structure and their substrate binding affinity which related to many diseases, such as melanoma, lumbar-disc herniation, type 2 diabetes, and pelvic organ prolapse (Ahluwalia et al., 2009; Cotignola et al., 2007; Hirose et al., 2008; Chen et al., 2010a). The GA genotype distribution in VSD group is much higher than control group.
Although there were few subjects of AA genotype in VSD group, there is an obvious difference in genotype distribution to control group.
Blankenberg et al. found that the G allele of MMP-9 R574P polymorphism was overrepresented in patients with histologically confirmed giant cell arteritis (GCA)
(Blankenberg et al., 2003); however, we do not find any significant differences in both the genotype and allele distribution between the VSD and control groups in Taiwanese population.
According to VSD patients different genotypes of MMP-9 Q279R and R574P polymorphism, there is no significant difference showed in MMP-9 activity detection, which indicates these SNPs may cause effect in protein structure and substrate binding efficiency but no notable effect in enzyme activity.
The VSD patients according to their serious level were further categorized to four groups.
All groups were conducted of the study that whether the serious level is correlative to MMPs activity. The most valuable find out in this research is that MMP-2 and MMP-9 enzyme activity increases with the defect serious level of VSDs increase (Figure 14). Indicating that
MMPs expression may affect the severe level of VSDs, and interfere the closure rate of VSDs.
The rate of spontaneous closure of VSDs has been reported to be between 11% and 70.8% in various researches (Kidd et al., 1993; Mehta and Chidambaram, 1992; Ahunbay et al., 1999; Eroğlu et al., 2003). In our study, spontaneous closure was detected in 23% of perimembranous defect (Table 7). Commonly, patient with small defect is considered more easy to happen spontaneous closure for not violent blood stream; but in our study, our samples showed that patients with severe defect are more possible to happen spontaneous closure, which the possibility of spontaneous closure are 38% and 14% for severe and slight VSD, respectively.
In several studies, it was proposed that ventricular septal aneurysm is an important mechanism of closure and shows a more favorable prognosis in perimembranous defects (Freedom et al., 1974; Ramaciotti et al., 1995). This study proposes that in vivo proteases are very possible relative to spontaneous closure of VSD. Figure 15 demonstrates that
spontaneous closure subgroup of VS group owns lower MMPs activity than the other
subgroup, and the results are opposite to VM and VL group, which their spontaneous closure subgroup own higher MMPs activity. Combined the results from Figure 14 and Figure 15, the reasons of VM and VL group have higher MMPs activity expressed in spontaneous closure subgroup are suspected to their severe defect level and the role of MMPs participating in spontaneous closure of VSD. However, the role of MMPs participating in spontaneous closure of VSD is advantage to closure or not still unknown. The further research of mechanism about MMPs in VSD closure is needed in the future.