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Synthesis and Validation of a Hydroxypyrone-Based, Potent, and Specific Matrix Metalloproteinase-12 Inhibitor with Anti-Inflammatory Activity In Vitro and In Vivo.

Aerts J, Vandenbroucke RE, Dera R, Balusu S, Van Wonterghem E, Moons L, Libert C, Dehaen W, Arckens L - Mediators Inflamm. (2015)

Bottom Line: The effects of inhibition of MMP-12 on endotoxemia and inflammation-induced blood-cerebrospinal fluid barrier (BCSFB) disruption were assessed both in vitro and in vivo.Moreover, an LPS-induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro.Taken together, this hydroxypyrone-based inhibitor is selective towards MMP-12 and displays anti-inflammatory activity in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroplasticity and Neuroproteomics, KU Leuven, Naamsestraat 59, 3000 Leuven, Belgium.

ABSTRACT
A hydroxypyrone-based matrix metalloproteinase (MMP) inhibitor was synthesized and assayed for its inhibitory capacity towards a panel of ten different MMPs. The compound exhibited selective inhibition towards MMP-12. The effects of inhibition of MMP-12 on endotoxemia and inflammation-induced blood-cerebrospinal fluid barrier (BCSFB) disruption were assessed both in vitro and in vivo. Similar to MMP-12 deficient mice, inhibitor-treated mice displayed significantly lower lipopolysaccharide- (LPS-) induced lethality compared to vehicle treated controls. Following LPS injection Mmp-12 mRNA expression was massively upregulated in choroid plexus tissue and a concomitant increase in BCSFB permeability was observed, which was restricted in inhibitor-treated mice. Moreover, an LPS-induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro. Taken together, this hydroxypyrone-based inhibitor is selective towards MMP-12 and displays anti-inflammatory activity in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus

In vivo and in vitro effect of MMP-12 inhibition in endotoxemia. (a) Survival in function of time of MMP-12+/+ (black) and MMP-12−/− (grey) mice following i.p. challenge with a lethal dose of LPS. (b) Survival in function of time of C57BL/6 mice injected with vehicle (black) or MMP-12i (grey) following i.p. challenge with a lethal dose of LPS. (c) Relative Mmp-12 expression in choroid plexus tissue isolated before and 1, 4, and 8 hours after LPS challenge (n = 3-4). (d) Relative permeability of the blood-CSF barrier determined by measuring leakage of fluorescently labeled dextran (4 kDa) from the blood into the CSF 8 hours after LPS challenge in vehicle (black) and MMP-12i (grey) treated wild type mice, compared to PBS injected wild type mice. (e) Normalized resistance measured at low frequency using the ECIS instrument of primary choroid plexus epithelial cells incubated with vehicle (black), 1000 ng/mL LPS (black dotted line), and 1000 ng/mL LPS pretreated with 1 μM MMP-12 inhibitor (grey) (n = 3). Data are presented as means ± standard error of mean (sem). Survival curves were compared using a log-rank test. Other data were analyzed by Student's t-test. ∗  0.01 ≤ P < 0.05; ∗∗  0.0001 ≤ P < 0.001; ∗∗∗∗P  < 0.0001.
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fig2: In vivo and in vitro effect of MMP-12 inhibition in endotoxemia. (a) Survival in function of time of MMP-12+/+ (black) and MMP-12−/− (grey) mice following i.p. challenge with a lethal dose of LPS. (b) Survival in function of time of C57BL/6 mice injected with vehicle (black) or MMP-12i (grey) following i.p. challenge with a lethal dose of LPS. (c) Relative Mmp-12 expression in choroid plexus tissue isolated before and 1, 4, and 8 hours after LPS challenge (n = 3-4). (d) Relative permeability of the blood-CSF barrier determined by measuring leakage of fluorescently labeled dextran (4 kDa) from the blood into the CSF 8 hours after LPS challenge in vehicle (black) and MMP-12i (grey) treated wild type mice, compared to PBS injected wild type mice. (e) Normalized resistance measured at low frequency using the ECIS instrument of primary choroid plexus epithelial cells incubated with vehicle (black), 1000 ng/mL LPS (black dotted line), and 1000 ng/mL LPS pretreated with 1 μM MMP-12 inhibitor (grey) (n = 3). Data are presented as means ± standard error of mean (sem). Survival curves were compared using a log-rank test. Other data were analyzed by Student's t-test. ∗  0.01 ≤ P < 0.05; ∗∗  0.0001 ≤ P < 0.001; ∗∗∗∗P  < 0.0001.

Mentions: Endotoxemia, that is, systemic administration of lipopolysaccharide (LPS), induces systemic inflammation and eventually lethality in mice. We have previously shown that mice can be protected from LPS-induced lethality by administration of a broad spectrum MMP inhibitor BB-94 [9] and several MMPs appeared to play a role in the LPS-induced lethality [8, 11–13]. Here, we studied the effect of MMP-12 deficiency on the LPS sensitivity by using MMP-12−/− mice. As shown in Figure 2(a), MMP-12−/− mice display a partial protection to an LD100 dose of LPS, indicating that MMP-12 plays a detrimental role in the LPS-induced lethality. To verify this, we treated wild type mice with the MMP-12i and challenged them with LPS (Figure 2(b)). MMP-12i treatment resulted in a significant reduction in LPS-induced lethality compared with vehicle treated mice, which again demonstrates that the compound most likely targets MMP-12. Systemic inflammation is known to be associated with loss of blood-brain barrier integrity and this has been correlated with lethality [29]. Next to the most studied endothelial blood-brain barrier (BBB), we have previously shown that the blood-cerebrospinal fluid barrier (BCSFB) which is formed by the choroid plexus epithelium (CPE) is also affected in response to systemic LPS administration [9]. mRNA expression analysis of choroid plexus tissue revealed 500-fold upregulation of Mmp-12 upon systemic LPS administration (Figure 2(c)), associated with an increase in BCSFB permeability (Figure 2(d)). Interestingly, the loss of BCSFB permeability was significantly reduced when endotoxic mice were treated with our MMP-12i (Figure 2(d)). Finally, we analyzed the effect of the MMP-12i on the BCSFB in vitro. Therefore, primary CPE cells were isolated and plated onto electrical cell impedance sensing (ECIS) arrays containing gold electrodes. ECIS is a real-time, label-free, impedance-based method used to study the barrier properties of cells grown in culture. After reaching confluency, cells were incubated with LPS or LPS supplemented with MMP-12i and compared to untreated cells. LPS resulted in a drop in normalized resistance compared to control and this was significantly reduced in the presence of MMP-12i (Figure 2(e)). These data clearly show that MMP-12 plays a role in the observed LPS-induced loss of BCSFB integrity.


Synthesis and Validation of a Hydroxypyrone-Based, Potent, and Specific Matrix Metalloproteinase-12 Inhibitor with Anti-Inflammatory Activity In Vitro and In Vivo.

Aerts J, Vandenbroucke RE, Dera R, Balusu S, Van Wonterghem E, Moons L, Libert C, Dehaen W, Arckens L - Mediators Inflamm. (2015)

In vivo and in vitro effect of MMP-12 inhibition in endotoxemia. (a) Survival in function of time of MMP-12+/+ (black) and MMP-12−/− (grey) mice following i.p. challenge with a lethal dose of LPS. (b) Survival in function of time of C57BL/6 mice injected with vehicle (black) or MMP-12i (grey) following i.p. challenge with a lethal dose of LPS. (c) Relative Mmp-12 expression in choroid plexus tissue isolated before and 1, 4, and 8 hours after LPS challenge (n = 3-4). (d) Relative permeability of the blood-CSF barrier determined by measuring leakage of fluorescently labeled dextran (4 kDa) from the blood into the CSF 8 hours after LPS challenge in vehicle (black) and MMP-12i (grey) treated wild type mice, compared to PBS injected wild type mice. (e) Normalized resistance measured at low frequency using the ECIS instrument of primary choroid plexus epithelial cells incubated with vehicle (black), 1000 ng/mL LPS (black dotted line), and 1000 ng/mL LPS pretreated with 1 μM MMP-12 inhibitor (grey) (n = 3). Data are presented as means ± standard error of mean (sem). Survival curves were compared using a log-rank test. Other data were analyzed by Student's t-test. ∗  0.01 ≤ P < 0.05; ∗∗  0.0001 ≤ P < 0.001; ∗∗∗∗P  < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: In vivo and in vitro effect of MMP-12 inhibition in endotoxemia. (a) Survival in function of time of MMP-12+/+ (black) and MMP-12−/− (grey) mice following i.p. challenge with a lethal dose of LPS. (b) Survival in function of time of C57BL/6 mice injected with vehicle (black) or MMP-12i (grey) following i.p. challenge with a lethal dose of LPS. (c) Relative Mmp-12 expression in choroid plexus tissue isolated before and 1, 4, and 8 hours after LPS challenge (n = 3-4). (d) Relative permeability of the blood-CSF barrier determined by measuring leakage of fluorescently labeled dextran (4 kDa) from the blood into the CSF 8 hours after LPS challenge in vehicle (black) and MMP-12i (grey) treated wild type mice, compared to PBS injected wild type mice. (e) Normalized resistance measured at low frequency using the ECIS instrument of primary choroid plexus epithelial cells incubated with vehicle (black), 1000 ng/mL LPS (black dotted line), and 1000 ng/mL LPS pretreated with 1 μM MMP-12 inhibitor (grey) (n = 3). Data are presented as means ± standard error of mean (sem). Survival curves were compared using a log-rank test. Other data were analyzed by Student's t-test. ∗  0.01 ≤ P < 0.05; ∗∗  0.0001 ≤ P < 0.001; ∗∗∗∗P  < 0.0001.
Mentions: Endotoxemia, that is, systemic administration of lipopolysaccharide (LPS), induces systemic inflammation and eventually lethality in mice. We have previously shown that mice can be protected from LPS-induced lethality by administration of a broad spectrum MMP inhibitor BB-94 [9] and several MMPs appeared to play a role in the LPS-induced lethality [8, 11–13]. Here, we studied the effect of MMP-12 deficiency on the LPS sensitivity by using MMP-12−/− mice. As shown in Figure 2(a), MMP-12−/− mice display a partial protection to an LD100 dose of LPS, indicating that MMP-12 plays a detrimental role in the LPS-induced lethality. To verify this, we treated wild type mice with the MMP-12i and challenged them with LPS (Figure 2(b)). MMP-12i treatment resulted in a significant reduction in LPS-induced lethality compared with vehicle treated mice, which again demonstrates that the compound most likely targets MMP-12. Systemic inflammation is known to be associated with loss of blood-brain barrier integrity and this has been correlated with lethality [29]. Next to the most studied endothelial blood-brain barrier (BBB), we have previously shown that the blood-cerebrospinal fluid barrier (BCSFB) which is formed by the choroid plexus epithelium (CPE) is also affected in response to systemic LPS administration [9]. mRNA expression analysis of choroid plexus tissue revealed 500-fold upregulation of Mmp-12 upon systemic LPS administration (Figure 2(c)), associated with an increase in BCSFB permeability (Figure 2(d)). Interestingly, the loss of BCSFB permeability was significantly reduced when endotoxic mice were treated with our MMP-12i (Figure 2(d)). Finally, we analyzed the effect of the MMP-12i on the BCSFB in vitro. Therefore, primary CPE cells were isolated and plated onto electrical cell impedance sensing (ECIS) arrays containing gold electrodes. ECIS is a real-time, label-free, impedance-based method used to study the barrier properties of cells grown in culture. After reaching confluency, cells were incubated with LPS or LPS supplemented with MMP-12i and compared to untreated cells. LPS resulted in a drop in normalized resistance compared to control and this was significantly reduced in the presence of MMP-12i (Figure 2(e)). These data clearly show that MMP-12 plays a role in the observed LPS-induced loss of BCSFB integrity.

Bottom Line: The effects of inhibition of MMP-12 on endotoxemia and inflammation-induced blood-cerebrospinal fluid barrier (BCSFB) disruption were assessed both in vitro and in vivo.Moreover, an LPS-induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro.Taken together, this hydroxypyrone-based inhibitor is selective towards MMP-12 and displays anti-inflammatory activity in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroplasticity and Neuroproteomics, KU Leuven, Naamsestraat 59, 3000 Leuven, Belgium.

ABSTRACT
A hydroxypyrone-based matrix metalloproteinase (MMP) inhibitor was synthesized and assayed for its inhibitory capacity towards a panel of ten different MMPs. The compound exhibited selective inhibition towards MMP-12. The effects of inhibition of MMP-12 on endotoxemia and inflammation-induced blood-cerebrospinal fluid barrier (BCSFB) disruption were assessed both in vitro and in vivo. Similar to MMP-12 deficient mice, inhibitor-treated mice displayed significantly lower lipopolysaccharide- (LPS-) induced lethality compared to vehicle treated controls. Following LPS injection Mmp-12 mRNA expression was massively upregulated in choroid plexus tissue and a concomitant increase in BCSFB permeability was observed, which was restricted in inhibitor-treated mice. Moreover, an LPS-induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro. Taken together, this hydroxypyrone-based inhibitor is selective towards MMP-12 and displays anti-inflammatory activity in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus