Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development

Abstract

Background: Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD).

Methods and findings: Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal.

Conclusions: MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.

Figure 1. Association between MMP16 polymorphisms (A: rs2664349 A/G; B: rs2664352 C/T) and the size of the activated fraction of MMP2 measured in tracheal aspirates.

Aspirates were collected immediately after birth (open bars), and between 1 and 3 days of life (closed bars). * p<0.05 in post-hoc analysis.

Figure 2. MMP16 immunoblots of tracheal aspirate supernatants from infants with the MMP16 AA-CC (lanes a–c) and GG-TT (lanes d–f) genotypes.

Upper: a 45-kDa band was clearly identified in AA-CC infants but was barely visible in GG-TT infants. Lower: densitometric analysis (arbitrary units [A.U.]) in 9 AA-CC infants and 8 GG-TT infants, showing a significant difference in the MMP16 level (Mann Whitney p<0.02).

Figure 3. Developmental pattern of MMP16 gene and protein expression in control lungs.

A. Immunoblot of lung homogenates from human fetuses without lung disease, at 11 to 36 weeks of gestation. B. Changes in the MMP16 mRNA expression level in rat whole lung tissue. Expression was quantified by real-time PCR from fetal life (canalicular stage of development) to adulthood, in 3 to 5 individual lung samples per stage. The birth level was arbitrarily given a value of 100. Values are means±SEM. * p<0.05 versus birth level. C. Immunoblot of lung homogenates from control rats, from fetal life to adulthood.

Figure 4. Changes in lung MMP16 mRNA expression in newborn rats exposed to insults associated with alveolar developmental arrest.

A: Exposure to hyperoxia (O₂>95%) from birth to day 6; B: Daily administration of dexamethasone from birth to day 5 (two doses). Results are expressed as a percentage of the control value. * p<0.05 in two-group comparisons.