Fig 2: NK2 homeobox 5 (Nkx2-5) regulates expression of MMPs and TIMPs in human monocyte-derived macrophages and human aortic smooth muscle cells. A, Representative immunoblot for MMPs and TIMPs in human peripheral blood monocyte-derived macrophages infected with Ad-EV or Ad-Nkx2-5. B, Representative immunoblot for MMPs and TIMPs in human aortic smooth muscle cells infected with Ad-EV or Ad-Nkx2-5. C, Quantification of band density in (A). Results are expressed as fold of control group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus Ad-EV group. D, Quantification of band density in (B). Results are expressed as fold of control group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus Ad-EV group. MMP indicates matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

Fig 3: NK2 homeobox 5 (Nkx2-5) directly regulates MMPs/TIMPs transcription. Luciferase (Luc) reporter constructs containing promoters of MMPs or TIMPs were cotransfected with an internal control plasmid pRL-TK into HEK293 cells, followed by infection with Ad-Nkx2-5 or Ad-EV. Schematic representation in the left panel demonstrates the range of promoters, and the numbers indicate nucleotide positions relative to the transcription initiation site. The right panel demonstrates relative luciferase activities, which are expressed as a percent of values determined in the Ad-EV-treated group. Data represent the mean±SEM of 3 independent experiments. *P<0.05 versus the Ad-EV-treated group. MMP indicates matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

Fig 4: Role of TNF-a, IL-1ß, and MIP-1a/CCL3 in nHZ-induced mRNA expression and protein release of human monocytic TIMP-1.Human adherent monocytes were left unfed (negative controls) or fed with nHZ (positive controls). Thereafter, cells were incubated for 15 h (mRNA studies, Panels A-B) or 24 h (protein studies, Panels C-D) in the presence/absence of the following treatments: single or combined doses (30 ng/ml) of anti-hTNF-a, anti-hIL-1ß and anti-hMIP-1a/CCL3 blocking antibodies (Panels A and C, blocking approach); and single or combined doses (20 ng/ml) of rhTNF-a, rhIL-1ß and rhMIP-1a/CCL3 (Panels B and D, mimicking approach). After lysis, TIMP-1 mRNA expression was measured by Real Time RT-PCR; alternatively, TIMP-1 protein levels in cell supernatants were evaluated by Western blotting and densitometry. Data are indicated as mean values+SEM or as a representative blot of three independent experiments. All data were evaluated for significance by ANOVA. Panel A: Vs unstimulated cells (column 1) *p<0.01; Vs untreated nHZ-fed cells (column 2) °p<0.05. Panel B: Vs unstimulated cells (column 1) *p<0.05, **p<0.01. Panel C: Vs unstimulated cells (column 1) *p<0.05, **p<0.0001; Vs untreated nHZ-fed cells (column 2) °p<0.01, °°p<0.0001; Vs anti-TNFa-treated nHZ-fed cells (column 3) §p<0.05. Panel D: Vs unstimulated cells (column 1) *p<0.05, **p<0.01, ***p<0.001.

Fig 5: Effect of matrix metalloproteinase-9 (MMP-9) on the tissue expression of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and cyclooxygenase-2 (COX-2). Rabbits in the MMP-9 group (n=10) were injected with MMP-9 at the 10th week. Rabbits in the control (Con; n=10) and chronic renal failure (CRF; n=10) groups were were injected with an equal volume of water. At the 14th week (the fourth week subsequent to the MMP-9 treatment), the animals were sacrificed and the kidneys were removed. Samples of kidney tissue were obtained and used as follows: (A) qPCR to examine the mRNA expression of MMP-9, TIMP-1 and COX-2; and (B) immunoblotting (IB) to examine the protein expression of MMP-9, TIMP-1 and COX-2. The representative bands in the IB from six rabbits in each group are shown. *P<0.05 vs. Con group; +P<0.05 vs. CRF group.