| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From the Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI.
Correspondence to: Robert M. Strieter, MD, FCCP, University of Michigan, Pulm/CCM, 6301 MSRB III, 1150 W Medical Ctr, Ann Arbor, MI 48109
Diffuse alveolar damage (DAD) of ARDS is a stereotypic response to a variety of etiologies.1 2 The pathologic findings of DAD follow an overlapping continuum of injury: acute exudative phase (approximately days 0 to 4); subacute proliferative phase (approximately days 2 to 8); and chronic fibrotic phase (approximately after day 4).2 3 4 This latter phase is associated with ARDS patients who require prolonged mechanical ventilation with both high inflation pressures and increased oxygen requirements.4 In addition, a significant proportion of ARDS survivors appear to have residual pulmonary fibrosis and compromised pulmonary function.5 6 7 This suggests that the pathogenesis of DAD that ultimately leads to the chronic fibrosis of ARDS demonstrates features of dysregulated repair with exaggerated intra-alveolar angiogenesis, fibroproliferation, and deposition of extracellular matrix, leading to progressive alveolar fibrosis and impaired lung function. Angiogenesis during DAD is not only a salient feature of the fibroproliferative response, but may represent a mechanism supporting mesenchymal cell proliferation and maintenance of intra-alveolar fibrosis. The mediators that orchestrate this aberrant neovascular response in DAD have not been fully elucidated. Members of the CXC chemokine family exert disparate effects in mediating angiogenesis as a function of the presence or absence of three amino acid residues (Glu-Leu-Arg; the `ELR' motif) in the NH2-terminus of these cytokines.8 CXC chemokines have potent angiogenic (ELR+) and angiostatic (ELR-) activity, respectively, and are important factors that regulate angiogenesis in both solid tumors and chronic fibroproliferative disorders.9 10
We hypothesized that for fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We postulated that an imbalance exists in the expression of angiogenic (KC and macrophage inflammatory protein [MIP]-2) vs angiostatic (interferon-inducible protein 10 [IP-10] and monokine induced by interferon-gamma [MIG]) CXC chemokines which favor net angiogenesis. To test this hypothesis, we designed a strategy to assess murine CXC chemokines in a murine model of cecal ligation and puncture (CLP) in the presence of hyperoxia, to simulate a clinical scenario of systemic sepsis and exposure to hyperoxia. CLP induces a polymicrobial peritonitis, which leads to systemic bacteremia and acute lung injury (ALI). This scenario is analogous to what occurs in humans with overt septic shock, ALI/ARDS with reduced PaO2/fraction of inspired oxygen (FIO2), and who are subsequently resuscitated and exposed to hyperoxia in the ICU.11
We first characterized our model by examining the survival of CD-1 mice after CLP. To assess whether differences exist when animals are exposed to hyperoxia (100% oxygen), as compared to room air under systemic sepsis, we performed CLP with a 21-gauge needle (approximately LD50 model under room air conditions) in 24 animals (12 in each group) and assessed survival. Oxygen or room air was delivered to the chambers under constant flow, and oxygen content of the chambers was monitored by an oxygen analyzer. At 24 h post-CLP, there were 8 of 12 (66%) and 5 of 12 (42%) animals alive in the presence of hyperoxia and room air, respectively, suggesting a survival advantage for animals undergoing CLP and exposure to hyperoxia. However, by 48 h, this survival advantage significantly changed with 4 of 12 (33%) and 5 of 12 (42%) animals surviving in hyperoxia and room air, respectively. The decline in survival in the hyperoxia group was apparent over the next 48 h, with only 1 of 12 (8%) animals surviving at 96 h in the hyperoxia-exposed group. There was no further decline in survival in the room air-exposed group over this same time period. These data support the contention that oxygen supplementation is important in mediating a survival advantage during the early resuscitative period; however, with continued hyperoxia (oxidant stress) exposure after initiation of sepsis, there is a significant disadvantage in survival.
To evaluate the difference in ALI under these conditions, four groups
of animals were studied in the following manner: CLP/hyperoxia;
CLP/room air; sham operated/hyperoxia; and sham operated/room air. CLP
was performed with a 21-gauge needle. The lungs of these animals were
evaluated under both light and transmission electron microscopy. The
lungs of animals that had undergone CLP/hyperoxia demonstrated
significant DAD with evidence of intra-alveolar neovascularization, as
well as early fibrosis with the presence of collagen fibrils. In
contrast, the ALI associated with CLP/room air demonstrated a reduced
injury pattern. The lung injury that was evident in
sham-operated/hyperoxia-exposed animals demonstrated a similar
pattern of injury as seen in the lungs of animals that had undergone
CLP/room air. The sham-operated/room air-exposed animal lungs appeared
to be morphologically normal. The lung injury severity by both
light and electron microscopy in this model system was
CLP/hyperoxia > CLP/room air 
sham operated/hyperoxia > sham
operated/room air. Moreover, the lung injury seen in the CLP/hyperoxia
animals was similar to that reported for humans at postmortem
associated with DAD of ARDS.2
3
4
Preliminary studies were next performed to quantitate changes in levels of specific cytokines of the lung under the above conditions at 8, 24, and 96 h. The lungs were processed as previously described and the cytokines measured using specific enzyme-linked immunosorbent assays (ELISAs). Both ELR+ (KC and MIP-2) and ELR- (IP-10 and MIG) CXC chemokines were significantly elevated at 24 h in both CLP groups, as compared with both sham-operated control groups. In contrast, by 96 h, a marked imbalance was appreciated in the presence of ELR+, as compared with ELR- CXC chemokines in animals that had undergone CLP/hyperoxia, as compared with the other groups. The ratio of ELR+:ELR- CXC chemokines was 21.5:1, 3.4:1, 3.2:1, and 0.8:1 for CLP/hyperoxia, CLP/room air, sham operated/hyperoxia, and sham operated/room air groups, respectively. These findings suggested a marked shift toward a predominance of angiogenic ELR+ CXC chemokines under conditions of CLP/hyperoxia.
To determine whether these changes in CXC chemokines paralleled angiogenic activity in the lung, we assessed the lung homogenates normalized to total protein in the CMP assay of neovascularization. Lungs of animals subjected to CLP and exposure to hyperoxia for 96 h demonstrated a marked neovascular response in the cornea. The neovascular response was CLP/hyperoxia > CLP/room air = sham operated/hyperoxia > sham operated/room air groups, respectively. These findings support the contention of elevated angiogenic activity that parallels evidence of increased pulmonary fibrosis.
To substantiate that these ELR+ (KC and MIP-2) CXC chemokines may be modulating lung tissue-derived angiogenic activity, we next assessed the in vivo angiogenic activity of CLP/hyperoxia lung tissue, in the presence or absence of control, neutralizing KC, or neutralizing MIP-2 antibodies, utilizing the CMP assay of neovascularization. Neutralizing antibodies to either KC (two of seven corneas; 29% positive) or MIP-2 (two of six corneas; 33% positive) markedly reduced the angiogenic activity from CLP/hyperoxia lung tissue (seven of eight corneas; 88% positive). Both KC and MIP-2 have been shown to be a potent chemotactic factors for neutrophils. However, their expression in the later phases in this model may not be necessarily associated with neutrophil extravasation in the lung for three reasons. First, the later peak in KC and MIP-2 expression at 72 to 96 h after CLP and continued exposure to hyperoxia is a time-frame after maximal neutrophil extravasation in this model. Second, the presence of neutrophils in various animal models of pulmonary fibrosis has actually been found to be beneficial and associated with a decrease in collagen levels, whereas, neutrophil depletion has been associated with augmented collagen synthesis and fibrosis.12 Thus, if KC and MIP-2 were playing a significant role in neutrophil recruitment at this later time point, then their levels would have correlated with both neutrophil infiltration, as well as reduced levels of collagen. Finally, the above findings support the notion that KC and MIP-2 are major angiogenic factors in the lung of CLP/hyperoxia animals, and at a phase that correlates with significant intra-alveolar granulation tissue formation of DAD.
Footnotes
This research was supported, in part, by the National Institutes of Health grant P50HL60289.
References
-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med 184,981-992This article has been cited by other articles:
|
|
 |
|
  M. D. Burdick, L. A. Murray, M. P. Keane, Y. Y. Xue, D. A. Zisman, J. A. Belperio, and R. M. Strieter CXCL11 Attenuates Bleomycin-induced Pulmonary Fibrosis via Inhibition of Vascular Remodeling Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 261 - 268. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
