New insights in extracellular matrix remodeling and collagen turnover related pathways in cultured human tenocytes after ciprofloxacin administration

Abstract

We characterized the effect of ciprofloxacin (CPX) in cultured human tenocytes by morphological and molecular methods. Collagen type I and III mRNA and protein levels were unaffected, but lysyl hydroxylase 2b mRNA levels progressively decreased after CPX administration. MMP-1 protein levels significantly increased after 20 μg/ml CPX administration but remained unmodified at the higher dose, whilst MMP-2 activity was unchanged. Tissue inhibitor of MMP (TIMP-1) gene expression decreased after CPX treatment, whilst TIMP-2 and transforming growth factor-β1 gene expression, the cytoskeleton arrangement, and cytochrome c expression remained unmodified. Secreted Protein Acidic and Rich in Cysteine mRNA and protein levels remained almost unchanged, whilst N-cadherin mRNA levels resulted significantly down-regulated and connexin 43 gene expression tended to decrease after CPX administration. The CPX-induced decreased ability to cross-link collagen and decreased TIMP-1 levels, possibly leading to higher activity of MMPs in ECM degradation, together with the down-regulation of N-cadherin and connexin 43 are consistent with a reduced ability to maintain tissue homeostasis, possibly making the tendon more susceptible to rupture.

Keywords: ciprofloxacin; collagen turnover; extra-cellular matrix remodelling; tendons; tenocytes.

Figure 1.

Bar graphs showing COL-I (a) and COL-III mRNA levels (b) assessed by real time PCR in untreated (CT) and CPX-treated tenocytes. Data were normalized on GAPDH gene expression. (c) Bar graphs displaying COL-I and COL-III (d) protein levels analyzed by slot blot in culture medium of CT and CPX treated tenocytes. Data are mean ± SD for two independent experiments for samples run in duplicate.

Figure 2.

Bar graphs showing mRNA levels for LH2b in CT and tenocytes treated with CPX at different doses as described in the Materials and methods section. Data were normalized on GAPDH gene expression and are expressed as mean ± SD for two independent experiments for samples run in duplicate.

Figure 3.

(a) Bar graphs showing MMP-1 protein levels analyzed by slot blot and MMP-2 activity assessed by SDS-zymography (b) in tenocyte serum-free conditioned media after densitometric analysis of immunoreactive and lytic bands, respectively. Data are expressed as densitometric units ± SD for two independent experiments for samples run in duplicate. (c) Bar graphs showing TIMP-1 and TIMP-2 (d) gene expression after normalization on GAPDH mRNA levels. Data are expressed as mean ± SD for two independent experiments for samples run in duplicate. *p<0.01 vs CT; ^p<0.05 vs CT; °p<0.05 vs 10 μg/ml

Figure 4.

Immunofluorescence analysis of the actin cytoskeleton in tenocytes untreated and treated with CPX. Representative immunofluorescence photomicrographs of microfilament distribution, evidenced by rhodamine-phalloidin labeling, in CT (a) and tenocytes after 10 μg/ml (b), 20 μg/ml (c) and 50 μg/ml CPX (d). DAPI was used for nuclear staining. Original magnification: 40×.

Figure 5.

Representative immunofluorescence analysis for vimentin intermediate filaments in CT (a) and tenocytes after 10 μg/ml (b), 20 μg/ml (c) and 50 μg/ml CPX (d). DAPI was used for nuclear staining. Original magnification: 40×.

Figure 6.

Representative immunofluorescence photomicrographs of microtubules in CT (a) and tenocytes after 10 μg/ml (b), 20 μg/ml (c) and 50 μg/ml CPX (d). DAPI was used for nuclear staining. Original magnification: 40×.

Figure 7.

Bar graphs showing SPARC gene expression (a) and protein levels in cell culture super-natants (b), N-cadherin (c) and CX43 (d) mRNA levels in tenocytes untreated (CT) and after CPX administration. Data were normalized on GAPDH mRNA levels and are expressed as mean ± SD for two independent experiments for samples run in duplicate. *P<0.05 vs CT; °P<0.01 vs CT

Figure 8.

Intrinsic apoptosis analysis. Representative microphotographs showing immunofluorescence detection of cytochrome c in CT (a) and 50 μg/ml CPX-treated (b) tenocytes. Untreated CT and CPX-treated cells showed a similar immunoreactivity characterized by a punctate cytoplasmic staining pattern typical for localization of cytochrome c into intact mitochondria. Original magnification 40×.