Introduction
The host response in sepsis is a dynamic process activating the pathways of coagulation, inflammation and tissue repair. When the response becomes overwhelming, it leads to multiple organ failure (MOF) and death [
1‐
3]. Disturbed connective tissue metabolism is the key element in complications of inflammatory disease, so it was of interest to determine whether high systemic inflammation in sepsis has any effect and whether the level of connective tissue metabolism reflects disease severity and outcome.
Fibroblasts synthesise a wide array of extracellular matrix proteins, predominantly type I and III collagens, which provide structural support to the organs [
4,
5]. The aim of this process is to maintain tissue integrity in a steady state and restore the integrity of the organ after injury. Prolonged inflammatory response may lead to persistent or progressive fibrosis impairing the function of an organ. Collagen synthesis has been shown to be pathologically increased, not only in wound keloids and wound infections, but also in acute respiratory distress syndrome (ARDS), chronic liver diseases, myocardial infarction and kidney diseases [
4,
6‐
10]. Indeed, it has been suggested that progressive fibrosis is a central mechanism of organ failure, which is related to the host's inflammatory responses and subsequent fibroblast response [
11].
In the course of collagen biosynthesis, procollagen-derived peptides are deposited in the extracellular matrix and released into the circulation. Aminoterminal propeptides are cleaved from procollagens in a one to one proportion and thus reflect the synthesis of collagen. Increased serum levels of procollagen type III propeptide have been found in severely injured patients and have been associated with MOF and death [
12]. Additionally, procollagen III propeptide levels in plasma and bronchoalveolar lavage fluid from patients with ARDS are increased in early phases and are related to disease progression, multiple organ dysfunction and death [
7,
13].
Cross-linked type I collagen telopeptides (ICTP) were assessed as markers of collagen I degradation. Previously, Wenisch and colleagues have reported elevated ICTP levels in Gram-negative septicaemia [
14].
As fibrosing activity, measured by synthesis and degradation of collagen, seems to have an important role in inflammatory processes, we hypothesised that procollagen propeptide serum levels have a prognostic value in MOF and death subsequent to severe sepsis. The collagen metabolism through the period of sepsis in humans has not been profoundly studied before.
Discussion
This is the first longitudinal study reporting serum procollagen propeptide levels in human severe sepsis. Previous studies have focused on collagen metabolism in severe trauma, ARDS or Gram-negative sepsis [
7,
12,
14]. Increasing collagen propeptide levels (PIIINP throughout the disease process and PINP in the late phase) were associated with the development of MOF and death and they correlated with maximum lactate concentrations. All the values in survivors had returned to the normal range and were lower at three and six months than they were at the beginning of the study.
Of the different organs, collagen synthesis in lungs has been most profoundly studied in critical illness. ARDS is the most severe manifestation of acute lung injury and is also one of the most common organ failures in severe sepsis. The collagen I and III propeptides have been showed to be elevated in plasma and bronchoalveolar lavage fluid in patients with ARDS during the first days of disease and are associated with increased risk of death [
7,
13,
19]. In our data the patients with lung specific SOFA scores of three to four had only slightly pronounced PINP, PIIINP and ICTP values (day one and the maximal values over the study period) compared with patients with less severe scores. The difference did not reach statistical significance (data not shown). Hence the increased procollagen propeptide levels observed in this study seem to be only partly due to increased synthesis and degradation of collagen in the lungs.
Waydhas and colleagues reported increased PIIINP serum concentrations in severely injured patients [
12]. Similar to our findings in septic patients, serum concentrations were elevated in severely injured non-survivors and in those who developed MOF. It was noted in the study by Waydhas and colleagues that PIIINP levels correlated with increasing bilirubin levels. The procollagen propeptides are eliminated by the liver, thus the increased serum levels may result from increased synthesis or decreased uptake by liver cells [
20]. The study by Waydhas and colleagues did not determine whether the increased concentrations were due to excess synthesis or diminished elimination [
12].
On the other hand, in alcoholic liver fibrosis it has been shown that elevated PIIINP concentrations are caused by increased histologically confirmed fibrogenesis [
21]. In our study, PINP and PIIINP did correlate with liver function implying that either synthesis or elimination by the liver in sepsis is affected. Because PINP and PIIINP are eliminated via the same liver endothelial cell receptor, the serum levels of both propeptides should have increased if the increased concentrations were solely a result of decreased elimination.
A correlation with kidney function was also observed. Small fractions of PIIINP are excreted by the kidneys [
22,
23]. Interestingly, increased PIIINP levels have been reported in acute renal disease, exemplifying the influence of systemic disease on collagen metabolism. Keller and colleagues reported that, compared with values in chronic renal failure, the values of PIIINP were even higher in patients with acute renal failure and MOF [
8]. Furthermore, experimental data have shown that renal damage increases the release of a collagen synthesis-stimulating factor [
24]. Previous data thus suggests that acute renal failure is associated with increased synthesis of type III collagen. In the present study, maximum PINP, PIIINP and ICTP levels did not have statistically significant prognostic values for liver and renal failure in the ROC analysis.
It is tempting to speculate that increased collagen propeptide levels found are at least partly due to increased synthesis and are likely to be a summation of collagen synthesis from different organs. To find out the contribution of the different organs affected, further studies are required.
ICTP is a marker of collagen degradation and is eliminated by the kidneys [
20]. In a small study Wenisch and colleagues reported elevated ICTP levels in Gram-negative sepsis on day 0 and day 28 [
14]. We found that serum ICTP, but not PINP, was increased in severe sepsis. Thus, the increased ICTP levels most likely indicate increased degradation of collagen type I. As type I collagen is most abundant in bone, it could be speculated that high levels of ICTP could partly be a result of immobilisation. However, increased ICTP levels most likely mirror high systemic inflammation, because the levels were highest in patients with the most severe forms of the disease. Collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells and inflammatory cells. MMPs are induced by proinflammatory cytokines (e.g. IL-1, IL-6 and TNF).
In vitro it has been shown that, following exposure to
S. aureus, fibroblasts have increased MMP expression, which is associated with degradation of collagen [
25].
Our study suggests that collagen turnover may be increased in severe sepsis. Over the past years, our understanding on the complexity of the host healing response in sepsis has grown: Phases of coagulation, inflammation and fibroproliferation overlap and exert regulatory control on one another. The collagen synthesis in fibroblasts is regulated by coagulation cascade proteases, proinflammatory cytokines and growth factors. Coagulation protease thrombin seems to act as fibroblast chemoattractant [
26], stimulator of procollagen production [
27], promoter of myofibroblast formation [
28] and MMP activator [
29]. Recently, a similar role of the upstream coagulation protease Xa has been acknowledged. It seems to enhance the expression of tranforming growth factor beta (TGF-β), fibroblast proliferation and differentiation to myofibroblasts, migration and fibronectin production [
30]. Thus the activated coagulation in sepsis is one factor promoting the fibrogenetic response.
Of the proinflammatory cytokines TNF-α has a pivotal effect on collagen synthesis. In addition to stimulating fibroblast growth and collagen synthesis, it has been shown that TNF-α in high concentrations inhibits collagen and fibronectin production and induces collagenase synthesis [
31]. Among the growth factors TGF-β deserves special attention. It is a multifunctional growth factor that regulates proliferation, differentiation of cells, protein synthesis and angiogenesis. TGF-β has been reported to act as an inducer, as well as an inhibitor, of fibroblast growth [
32]. Increased fibrosis is mediated by TGF-β1 in various disease states, and progressive fibrosis has been suggested to be a common pathway to organ failure [
11]. Accordingly, in ARDS it has been demonstrated that bronchoalveolar lavage fluid obtained from patients is capable of activating a human procollagen 1 promoter by means of TGF-β1 present in the bronchoalveolar lavage fluid. Furthermore, in ARDS TGF-β1 levels have been shown to be higher in non-survivors, although the result is not statistically significant [
33]. Higher levels have also been reported in trauma patients developing sepsis [
34]. Indeed, sepsis could be called a systemic wound with activated coagulation, inflammation and fibrogenetic response.
Other factors that can affect collagen metabolism in severe sepsis include surgery, hydrocortisone treatment and tissue hypoxia. Surgery and trauma induce the healing process and thus account for the fibroproliferative response. In a previous study, it was shown that surgery itself (and wound infection especially) increases serum procollagen concentrations [
35]. In our study no differences could be found between the surgical and medical groups. The surgical group consisted of patients with trauma or those who underwent major surgical procedure requiring general anaesthesia. Minor standard ICU procedures such as tracheostomy, drainage or cannulations were also performed in the medical group and could partly have contributed to the controversial result of our study.
It is known that corticosteroid therapy reduces collagen deposition [
7,
36]. In our material, treatment of sepsis with steroids decreased serum PINP levels, indicating that type I collagen synthesis is decreased in the early phase (up to six days) of sepsis in patients treated with hydrocortisone. After hydrocortisone therapy, which most often lasted seven days, the PINP values were upregulated as in the group not treated with hydrocortisone. Hypoxia is a fibrotic stimulus associated with enhanced collagen synthesis and it has been shown to augment collagen prolyl 4-hydroxylase activity
in vitro [
37]. Tissue hypoxia and activation of the coagulation and inflammatory cascades play a key role in the pathogenesis of MODS. Although adequate initial resuscitation usually restores oxygen delivery at the systemic level, regional hypoxia at the organ level is a well-documented phenomenon. The mechanisms are considered to include microcirculatory disturbances, that block the oxygen supply, and mitochondrial malfunction that results in inadequate use of oxygen at the cellular level. Increased circulating lactate levels are suggestive of tissue hypoxia and are associated with a poor outcome. In our study PINP, PIIINP and ICTP correlated with maximum lactate levels. The importance of tissue hypoxia in the stimulation of collagen synthesis is also suggested by the results in patients with chronic heart failure in which relative collagen deposition in the intestinal wall was the highest in advanced cases of heart failure [
38]. Furthermore, in a rat model, sepsis has been shown to induce significant increases in collagen content in hepatic and ileal interstitial tissues, which were prevented with a leucotriene antagonist [
39]. Yet there is also evidence to the contrary. In a mice model of lipopolysaccharide-stimulated ARDS, hypoxia suppressed inflammation in lungs via adenosine A
2A-receptor-mediated pathway and resulted in lower lung injury score and thickening of the alveocapillary membrane [
40].
This study is limited by the fact that our study population was relatively small because this was a one-centre study and a considerable number of patients were excluded because of underlying diseases affecting collagen metabolism. Second, the controls were healthy volunteers and thus could not be matched for chronic diseases, of which arteriosclerosis, diabetes and pulmonary diseases may have altered collagen metabolism. Third, the serum markers of inflammation were not measured. The septic response is individual and patients may have entered the study in different phases of inflammation, although all of them entered within 48 hours of the first organ failure. Further studies are needed to connect the levels of collagen turnover to timely development of coagulation and inflammatory responses. Nonetheless, this study provides new in vivo measured information on connective tissue metabolism and its timely development in sepsis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors participated in the study design. FG participated in collecting the data, performed statistical analysis and drafted the manuscript with TA. MK participated in collecting the data. VK conceived the study and helped to draft the manuscript. AO provided the equipment for the suction blister method and helped to draft the manuscript. JR provided collagen propeptide analyses. JL helped to draft the manuscript. JS conceived the study with VK. TA performed the statistical analysis and drafted the manuscript with FG. All authors read and approved the final manuscript.