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Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK, Schuchman EH, Fuks Z, Kolesnick R - J. Exp. Med. (1997)

Bottom Line: TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses.Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha.These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

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Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C57BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an  intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in  Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C57BL/6 treated as in A.  The number in parenthesis indicates the number of animals in each group.
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Figure 6: Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C57BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C57BL/6 treated as in A. The number in parenthesis indicates the number of animals in each group.

Mentions: To provide additional support for the notion that endothelial damage is essential for evolution of the endotoxic response, C57BL/6 mice were treated concomitantly with LPS and bFGF. Prior studies from our laboratory showed that bFGF protected endothelium in vitro and in vivo from radiation-induced apoptosis (31). In vivo, intravenously injected bFGF has been shown to be retained within blood vessels, apparently bound to the heparan sulfate proteoglycan coating the vascular surface of the endothelium and its basement membrane (31). Consequently, intravenously injected bFGF served as a selective endothelial survival factor, preventing radiation-induced apoptosis and lethal radiation pneumonitis (36). Fig. 6 A shows that bFGF abrogated LPS-induced apoptosis in the endothelium of the intestine and lung of C57BL/6 mice. Fig. 6 B demonstrates that intravenous bFGF, when injected concomitantly with a dose of 175 μg of LPS/25g mouse, provided protection from the lethal effects of LPS (P <0.001 vs. untreated). bFGF also rescued C57BL/6 mice from maximal doses of 270 and 350 μg of LPS/25g mouse, although the protection was not as complete (data not shown). Additional studies delineated the site of bFGF action. Table 2 shows that while TNF-α was not detected in the serum of sham- or bFGF-injected animals, 175 μg of LPS/25g mouse induced an elevation of serum TNF-α to a maximum of 4.2 ± 0.9 ng/ ml. The LPS-induced elevation of serum TNF-α was not blocked by bFGF. In contrast, bFGF prevented the elevation of tissue ceramide in response to LPS. These studies indicate that intravenous bFGF does not affect cell types that generate TNF-α in response to LPS (i.e., monocytes and macrophages), but specifically targets endothelial cells and the ceramide response to TNF stimulation. These data also substantiate endothelial damage as mandatory for LPS-induced death, and define inhibition of TNF signaling as the mechanism of the protective effect of bFGF on endothelium.


Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK, Schuchman EH, Fuks Z, Kolesnick R - J. Exp. Med. (1997)

Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C57BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an  intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in  Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C57BL/6 treated as in A.  The number in parenthesis indicates the number of animals in each group.
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Figure 6: Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C57BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C57BL/6 treated as in A. The number in parenthesis indicates the number of animals in each group.
Mentions: To provide additional support for the notion that endothelial damage is essential for evolution of the endotoxic response, C57BL/6 mice were treated concomitantly with LPS and bFGF. Prior studies from our laboratory showed that bFGF protected endothelium in vitro and in vivo from radiation-induced apoptosis (31). In vivo, intravenously injected bFGF has been shown to be retained within blood vessels, apparently bound to the heparan sulfate proteoglycan coating the vascular surface of the endothelium and its basement membrane (31). Consequently, intravenously injected bFGF served as a selective endothelial survival factor, preventing radiation-induced apoptosis and lethal radiation pneumonitis (36). Fig. 6 A shows that bFGF abrogated LPS-induced apoptosis in the endothelium of the intestine and lung of C57BL/6 mice. Fig. 6 B demonstrates that intravenous bFGF, when injected concomitantly with a dose of 175 μg of LPS/25g mouse, provided protection from the lethal effects of LPS (P <0.001 vs. untreated). bFGF also rescued C57BL/6 mice from maximal doses of 270 and 350 μg of LPS/25g mouse, although the protection was not as complete (data not shown). Additional studies delineated the site of bFGF action. Table 2 shows that while TNF-α was not detected in the serum of sham- or bFGF-injected animals, 175 μg of LPS/25g mouse induced an elevation of serum TNF-α to a maximum of 4.2 ± 0.9 ng/ ml. The LPS-induced elevation of serum TNF-α was not blocked by bFGF. In contrast, bFGF prevented the elevation of tissue ceramide in response to LPS. These studies indicate that intravenous bFGF does not affect cell types that generate TNF-α in response to LPS (i.e., monocytes and macrophages), but specifically targets endothelial cells and the ceramide response to TNF stimulation. These data also substantiate endothelial damage as mandatory for LPS-induced death, and define inhibition of TNF signaling as the mechanism of the protective effect of bFGF on endothelium.

Bottom Line: TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses.Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha.These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

Show MeSH
Related in: MedlinePlus