2.1. MARCO TEORICO
2.1.14. SUPERINTENDENCIA DE ADUANAS Y DE ADMINISTRACIÓN
Research into the pathogenesis of bacterial sepsis has increased dram atically
over the last decade. It is now clear that the host response to bacterial infection
is at least if n o t m ore im portant than the invading organism in dictating the
outcom e of patients w ith septic shock (Bone 1991, Cohen et al 1991, Glauser et
al 1991, Glauser et al 1994). Three areas of investigation have been particularly
influential in directing the work presented in this thesis. 1) The im portance of
vascular endothelial cells in the pathophysiology of sepsis (Levin 1990, Glauser
et al 1994); 2) The em erging role of GAGs in a m u ltitu d e of physiological
processes w hich m ay be affected in inflam m atory conditions (Lindahl et al
1978, Kjellén et al 1991b, H ardingham et al 1992)an d 3) The potentially
dam aging effects of cytokines and neutrophils on host tissues (Smedley et al
1986, W estlin et al 1993). This thesis has attem pted to fuse these three areas of
research and provide a firm basis for future studies directed at establishing the
role of endothelial GAGs in bacterial sepsis.
At the beginning of this project it became ap p aren t th at cu rren t m ethods of
investigating endothelial GAGs w ere not ideally suited for this study. The
com bination of an inadequate supply of umbilical cords and the prohibitive
costs of cytokines and w ere particularly problematic. In chapter 3, a range of
m eth o d s w ere th erefo re dev elo p ed for in v estig atin g en d o th e lia l GAG
m etabolism in com paratively sm all sam ples of c u ltu re d HUVEC and
supernatants. A reliable m ethod of extracting GAGs based on alcian blue
precipitation w as validated and chosen for subsequent studies. The use of
specific glycanases and nitrous acid w ith cellulose acetate electrophoresis w as
show n to be effective in identifying the nature of extracted GAGs and found to
Deficiencies w ith these techniques were how ever recognised and detailed in
the conclusions to chapter 3.
Endothelial cell GAG m etabolism in both unstim ulated and cytokine treated
HUVEC cu ltu res w ere th en investigated u sing the m ethods described in
chapter 3. U nstim ulated HUVEC were show n to release HS, DS and CS into the
culture m edium at increasing levels until the cultures becam e confluent. A
significant p roportion of these GAGs were found to be new ly synthesised as
determ ined by incorporation into the extracted GAGs. O nly HS and DS
were detected in the cell layer and in m uch sm aller quantities (< 20%) of that
found in culture supernatants.
TNF, IL l an d IFN - gam m a all had a profound effect on HUVEC GAG
metabolism. TNF and ILl inhibited GAG synthesis and caused the release of
preform ed GAGs from the cell layer. In contrast, IFN - gam m a stim ulated both
the synthesis and release of all sulphated GAGs in the endothelial cultures.
E ndotoxin an d IL6 w ere not found to have a significant effect on GAG
m etabolism in this model. The com bination of endotoxin, IL l or TNF w ith
separated neutrophils caused a dram atic reduction in cell associated GAGs.
This w as also associated w ith increased levels in cu ltu re su p e rn ata n ts
suggesting th at neutrophil derived proteases and or glycanases m ay have been
responsible. The m echanism s of GAG m odulation described in chapter 4 will
require further investigation and will form the basis of future studies.
Follow ing on from these findings, a histochem ical m eth o d of detecting
sulphated GAGs associated w ith the endothelial cells was developed (chapter
5). Extensive m an ip u latio n of pH was required to obtain the necessary
specificity of GAG staining required for this study. HS and DS w ere confirmed
identified. The im portance of methodological considerations in histochemical
an aly sis w as h ig h lig h ted w ith the d e m o n stra tio n of v astly different
appearances in m ethanol and aldehyde fixed cells. How ever, experiments with
cytokines alone and w ith ILl, TNF or endotoxin and neutrophils, corroborated
the biochemical data presented in chapter 4, irrespective of the fixation methods
employed.
The results from chapters 4 and 5 prom pted an investigation into the conditions
required for inducing endothelial injury. A m odel of endothelial injury in
w hich dam age was assessed using a combination of GAG histochem istry and
fibronectin staining w as developed (chapter 6). This w as com bined w ith
sim ultaneous analysis of neutrophil activation using flow cytometric analysis
of the adhesion molecules GD IS/G DI lb and L-selectin. Loss of GAGs from the
endothelial cells w as found to be a very sensitive m arker of endothelial injury
occurring in all of the conditions used in the study. In contrast fibronectin
staining w as only dim inished w hen both endotoxin and fMLP w ere added to
the cu ltu red cells. Sim ultaneous analysis of neu tro p h il activation by flow
cytom etry dem onstrated that both endothelial and neutrophil activation were
required to cause maximal damage in this model of endothelial injury.
Specific questions have been posed at the end of each chapter of this thesis and
will form the basis of future studies. However, tw o im portant general questions
will be addressed below:
1) H ow valid are the findings presented in this thesis ?