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Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis.

Yan JT, Wang T, Wang DW - Acta Pharmacol. Sin. (2009)

Bottom Line: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney.Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis.The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine and the Institute of Hypertension, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-mediated HK gene therapy protects against organ damage by inhibiting cell apoptosis.

Methods: rAAV encoding HK (rAAV-HK) or LacZ (rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells.

Results: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apoptosis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT.

Conclusion: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

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Related in: MedlinePlus

The kallikrein/kinin system reduces the ratio of apoptotic-to-live cells after apoptosis induction. HEK 293 cells were examined under a fluorescent microscope after acridine orange/ethidium bromide staining (A). Bright green cells with condensed chromatin are live apoptotic cells. Bright orange cells with condensed chromatin are dead apoptotic cells. Fainter green and orange cells with diffuse chromatin are non-apoptotic live and dead cells, respectively. The results showed that apoptosis increased when cells were exposed to LPS in non-FBS medium (positive control group) compared to cells in the same medium without LPS (negative control group). Treatment with kinin or rAAV-HK markedly reduced LPS-induced HEK 293 apoptosis (P<0.05), while rAAV-lacZ had no effect on apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the effects of kinin on apoptosis(B). cP<0.01 vs rAAV-LacZ.
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fig4: The kallikrein/kinin system reduces the ratio of apoptotic-to-live cells after apoptosis induction. HEK 293 cells were examined under a fluorescent microscope after acridine orange/ethidium bromide staining (A). Bright green cells with condensed chromatin are live apoptotic cells. Bright orange cells with condensed chromatin are dead apoptotic cells. Fainter green and orange cells with diffuse chromatin are non-apoptotic live and dead cells, respectively. The results showed that apoptosis increased when cells were exposed to LPS in non-FBS medium (positive control group) compared to cells in the same medium without LPS (negative control group). Treatment with kinin or rAAV-HK markedly reduced LPS-induced HEK 293 apoptosis (P<0.05), while rAAV-lacZ had no effect on apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the effects of kinin on apoptosis(B). cP<0.01 vs rAAV-LacZ.

Mentions: Having determined the protective effects of kallikrein against apoptosis in vivo, we then sought to confirm these effects in an in vitro cell culture system. The effects of the kallikrein/kinin system on HEK 293 cell apoptosis were detected by acridine orange/ethidium bromide staining and flow cytometry (double staining with annexin V and PI). rAAV-HK and Kinin treated HEK 293 cells had markedly reduced LPS-induced apoptosis (P<0.05), while rAAV-lacZ had no effect on LPS-induced apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the anti-apoptotic effect of kinin (Figures 4, 5). These data indicate that the kallikrein/kinin system has a direct anti-apoptotic effect by activating the B2 receptor.


Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis.

Yan JT, Wang T, Wang DW - Acta Pharmacol. Sin. (2009)

The kallikrein/kinin system reduces the ratio of apoptotic-to-live cells after apoptosis induction. HEK 293 cells were examined under a fluorescent microscope after acridine orange/ethidium bromide staining (A). Bright green cells with condensed chromatin are live apoptotic cells. Bright orange cells with condensed chromatin are dead apoptotic cells. Fainter green and orange cells with diffuse chromatin are non-apoptotic live and dead cells, respectively. The results showed that apoptosis increased when cells were exposed to LPS in non-FBS medium (positive control group) compared to cells in the same medium without LPS (negative control group). Treatment with kinin or rAAV-HK markedly reduced LPS-induced HEK 293 apoptosis (P<0.05), while rAAV-lacZ had no effect on apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the effects of kinin on apoptosis(B). cP<0.01 vs rAAV-LacZ.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4007182&req=5

fig4: The kallikrein/kinin system reduces the ratio of apoptotic-to-live cells after apoptosis induction. HEK 293 cells were examined under a fluorescent microscope after acridine orange/ethidium bromide staining (A). Bright green cells with condensed chromatin are live apoptotic cells. Bright orange cells with condensed chromatin are dead apoptotic cells. Fainter green and orange cells with diffuse chromatin are non-apoptotic live and dead cells, respectively. The results showed that apoptosis increased when cells were exposed to LPS in non-FBS medium (positive control group) compared to cells in the same medium without LPS (negative control group). Treatment with kinin or rAAV-HK markedly reduced LPS-induced HEK 293 apoptosis (P<0.05), while rAAV-lacZ had no effect on apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the effects of kinin on apoptosis(B). cP<0.01 vs rAAV-LacZ.
Mentions: Having determined the protective effects of kallikrein against apoptosis in vivo, we then sought to confirm these effects in an in vitro cell culture system. The effects of the kallikrein/kinin system on HEK 293 cell apoptosis were detected by acridine orange/ethidium bromide staining and flow cytometry (double staining with annexin V and PI). rAAV-HK and Kinin treated HEK 293 cells had markedly reduced LPS-induced apoptosis (P<0.05), while rAAV-lacZ had no effect on LPS-induced apoptosis. Hoe 140, a B2 receptor inhibitor, attenuated the anti-apoptotic effect of kinin (Figures 4, 5). These data indicate that the kallikrein/kinin system has a direct anti-apoptotic effect by activating the B2 receptor.

Bottom Line: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney.Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis.The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine and the Institute of Hypertension, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-mediated HK gene therapy protects against organ damage by inhibiting cell apoptosis.

Methods: rAAV encoding HK (rAAV-HK) or LacZ (rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells.

Results: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apoptosis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT.

Conclusion: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

Show MeSH
Related in: MedlinePlus