Appendix
UNIVERSITY OF BRISTOL
Bristol Heart Institute
Work in progress
Role of NF-κB and PKC-ζ in endothelial production of
MMPs
Tutor:
Supervisor:
10.1 BACKGROUND
Vascular endothelium is a dynamic cellular interface between the vessel wall and the bloodstream that regulates the physiological effects of humoral and biomechanical stimuli. Inflammatory cytokines and hemodynamic forces (such as blood pressure, wall tension, and shear stress) can cause weakening of the vessel wall. Extracellular matrix (ECM) macromolecules are represented by collagens types I, III, IV, and V, elastin,proteoglycans, and glycoproteins and are important for creating the cellular environments required during development and morphogenesis. The matrix rather than being merely a systemof scaffolding for the surrounding cells, is a dynamic structure that is central to the control of vascular remodelling 1.
Connectivetissue repair and remodelling to maintain matrix integrity involve the synthesis and removal of matrix proteins, a process that depends on the action of a multeplicity of proteases and their inhibitors 2. Matrix metalloproteinases (MMPs), collectively called matrixins, represent the main class involved in ECM degradation. Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous tissue inhibitors (TIMPs) 3,4. A loss of activity control may result in diseases such as atherosclerosis, arthritis, cancer, aneurysms, nephritis, tissue ulcers, and fibrosis. Atherosclerosis is a major cause of coronary heart disease, and MMPs enhanced expression in the atherosclerotic lesion contribute to weakening of the vascular wall by degrading the ECM, which results in cardiovascular remodelling 5. Endothelial cells (ECs), the sensors of increased flow and wall shear stress, play an important role in vascular remodelling through the release of MMPs 1.
MMPs are a family of Zn2+-containing and Ca2+-dependent enzymes that are either secreted from the cell or anchored to the plasma membrane. On the basis of substrate specificity, sequence similarity, and domain organization, MMPs can be divided into six groups:
collagenases, gelatinases, stromelysins, matrilysins, membrane typed MMPs, other MMPs 4. The first target for remodelling by ECs is the basement membrane and MMP-2 and MMP-9 appear specialised for this function; they belong to gelatinases group and readily digest the denatured collagens, gelatins and also plays an important role in angiogenesis 2,6. Like most MMPs, MMP-2, secreted as an inactive proenzyme, requires proteolytic removal of a terminal propeptide domain for its activation. Cleavage and activation of MMP-2 is achieved by a membrane type 1 MMP (also known as MMP-14) 7. MMP-14 and -2 expression and activation have been associated with neointimal growth after mechanical injury to the vascular wall 8. Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-α (TNF-α) and mechanical stretch have been shown to induce expression and activation of MMP in ECs 9-12. Little is known about the signal pathway mediating the stretch-induced MMP expression 13.
Understanding the factors responsible for upregulating MMP-2, MMP-9 and MMP-14 might lead to the development of inhibitors of adverse remodelling, for example in vulnerable atherosclerotic plaques, in arteriovenous fistulas and in the micovasculature in response to hypertension 14. Previous studies showed that activation of nuclear factor kappa (NF-kB) and proteine kinase C-zeta (PKC-ζ) are involved in MMP regulation pathway 15-18.
Atherosclerosis is a focal inflammatory disease that develops preferentially in vascular areas exposed to low and disturbed flow within the vasculature (such as bifurcations or curvatures)
19
. In contrast, straight vascular segments that experience unidirectional flow appear relatively resistant to atherogenesis 20. Many findings suggest that some of these protective effects of flow are mediated via inhibition TNF-α signaling in ECs 21,22. TNF-α is a proinflammatory cytokine, produced by activated leukocytes and ECs during vascular injury 23. Binding of TNF-α to the TNF receptor-1 (TNFR1) is followed by a rapid transcription of cell surface adhesion molecules (CAM) such as intercellular cell adhesion molecule-1 (ICAM-1) 24 as well as induction of apoptosis 25 in vivo and in vitro 26. Previous reports from our group and
others showed that unidirectional flow, via inhibition of cytokine-mediated signaling 27,28 prevents EC apoptosis 29,30 and CAM expression 31. However, molecular and signaling events governing this interplay between flow and cytokine signaling are not fully understood. PKC enzymes are serine/threonine kinases that phosphorylate several effectors proteins in a celland stimulus-specific manner 32. Among the PKC family, the atypical PKC-ζ has recently emerged as an important isoform in ECs. PKC-ζ, promotes the adhesive phenotype of ECs via the regulation of NFkB-dependent ICAM-1 expression 24,33. A recent study demonstrated a correlation between PKC-ζ activity and flow pattern in pig arteries, with lower PKC-ζ activity in ECs exposed to unidirectional flow compared with disturbed flow 34. Prolonged exposure to disturbed flow induces CAM expression in ECs in vitro and in vivo 35. Together, these observations suggest that PKC-ζ activity, differentially regulated by flow versus TNF-α, could be an important determinant of atherogenesis susceptibility via regulation of inflammatory pathways.
10.2 SPECIFIC AIM AND INNOVATIVE ASPECTS
The present study was designed to investigate the role of NF-kB and PKC-ζ on cytokine-induced and flow-mediated MMPs increase in ECs. We aimed to demonstrate an involvement of NF-kB and PKC-ζ in the upregolation of MMP-2, MMP-9 and MMP-14 in human umbilical vein endothelial cells (HUVECs) exposed to TNF-α or different shear stress condition. If so, overexpression of the inhibitory form of NF-κB and PKC-ζ might reduce MMPs production and specific inhibitors of PKC-ζ could be attractive agents to target adverse remodelling.
The results of this study might allow a deeper insight into the mechanisms of activation/inhibition of NF-kB and PKC-ζ and therefore of production/inhibition of MMP, important processes involved in vascular remodelling. This study might supply the justification for a subsequent in vivo study in PKC- ζ knockout mice.
10.3 METHODS
10.3.1 HUVEC cell culture
HUVECs 36 were cultured under different shear stress conditions using a custom-built flow apparatus or treated with TNF- α. We wanted to mimic the flow in different area of atherosclerotic artery in comparison with healthy artery. We reproduced in vitro physiological flow condition by normal laminar shear stress and the pathological one by high laminar shear stress, where the vessel is narrow because of the plaque and oscillatory shear stress where the flow is turbulent at the shoulder of the plaque.
10.3.2 Gelatin zymography
This technique 37 allows detecting gelatinase activity and so MMPs upregulation in cultured conditioned media.
Briefly, 20µl aliquots of conditioned media were mixed with an equal volume of non-reducing Laemmli sample buffer and electrophoresed at 4 °C in 7.5% SDS-polyacrylamide gels containing 2 mg/ml gelatin derived from porcine skin collagen (Sigma). After electrophoresis, the gels were washed clear of SDS by incubating for 1 h with two changes of 2.5% (v/v) Triton X-100. Gels were then incubated overnight in substrate buffer (50 mM Tris, pH 7.6, 50 mM NaCl, 10 mM CaCl2, and 0.1% Triton X-100) at 37 °C. The gels were then
stained with 0.1% Coomassie Brilliant Blue, and gelatinolytic activity was revealed as clear bands against a blue-stained background.
10.3.3 Semiquantitative and quantitative RT-PCR
We designed primers for vascular cell adhesion molecule(VCAM)-1, endothelial nitric oxide synthase(eNOS), heme oxygenase(HO)-1, matrix metalloproteinase(MMP)-9, 7, MMP-10, kruppel like factor(KLF)-2, some component of NADPH oxidase such as NOX-1, NOX-2 or gp91phox, NOX-4, NOX-5 and p47phox. We performed gradient RT-PCR to establish the
right annealing temperature and cycle number. We performed quantitative RT-PCR 38 to measure relative expression of genes in cDNA samples from HUVECs under different flow conditions.
Briefly, HUVECs were cultured on glass slides (2x105 cells per slide) previously coated with 0.2% gelatine and exposed for 24h to different flow conditions. Total mRNA was extracted by using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions.
Reverse transcription (RT)–polymerase chain reaction (PCR) was performed with Taq polymerase (Qiagen) using primers for different genes involved in “remodelling” (VCAM-1, MMP-9, eNOS etc) and GAPDH for various numbers of cycles to ensure that reactions did not reach saturation. Products were separated on agarose gels; results were expressed in arbitrary units and adjusted for GAPDH mRNA levels.
Real time (RT)–PCR was performed using a LightCycler (Roche) with QuantiTect SYBR Green PCR system (Qiagen) using primers for different genes involved in “remodelling” and 18S ribosomal RNA. Results were expressed in fold increase and adjusted for 18s mRNA levels.
10.3.4 Western Blot
This technique allows quantification of protein expression using specific antibodies.
Briefly, 10-20 µg of proteins were denatured by the addition of a reducing sample loading buffer containing β-mercaptoethanol and then heat denatured at 100 °C for 5 min. Samples were then electrophoresed at 95 V at 4 °C in a discontinuous buffer system comprising an 8% (w/v) polyacrylamide resolving gel and a 4% (w/v) polyacrylamide stacking gel. Samples were calibrated with a high molecular weight marker (Sigma). After electrophoresis, the gels were removed and equilibrated in transfer buffer (192 mM glycine, 25 mM Tris-HCl, pH 8.3, 20% (v/v) methanol), following which the protein samples were transferred onto Hybond-C nitrocellulose pure membrane (Amersham Biosciences), at 300 mA for 3 h at 4 °C. After
transfer, nonspecific binding sites were blocked by incubating in “blocking buffer” TBS-T-M (1xTBS (20 mM Tris, 137 mM NaCl), 5% skim milk powder (Marvel), 0.1% Tween 20) and then incubated overnight in the primary antibody at 4 °C. After incubation, the membranes underwent several washes in the blocking buffer, after which they were incubated for 1 h at room temperature with the peroxidase-conjugated secondary antibody. Unbound secondary was removed by washing in TBS-T. Detection was achieved with the ECL Western blotting kit with exposure to Hyperfilm x-ray film (Amersham Biosciences).
10.3.5 Use of adenoviral transduction for overexpression of Dominant Negative (DN) PKC Virus
An effective kinase-defective DN mutant derivative of PKC was engineered into a tetracycline-regulated binary adenoviral system. The DN-PKC cDNA was cloned under the control of the tetracycline response element. Expression of this transgene is dependent upon the presence of a second virus expressing the Tet-OFF transcriptional regulator under constitutive (cytomegalovirus) control. Following the application of the tetracycline analogue doxycycline, the Tet-OFF molecule can no longer bind and transcription of the PKC transgene ceases 39,40. HUVECs were infected with adenoviruses that drive overexpression of dominant negative PKC-ζ under tetracycline and constitutive expression to determine its role. Briefly, HUVECs (~3x105 cells/well) were seeded in 6-well plates. On the following day, cells were infected with the DN-PKC and Tet-OFF viruses at a multiplicity of infection of 300 and incubated for 16 h. This was followed by an additional 24h incubation in complete medium with or without 1 mg/ml doxycycline so as to allow for viral transgene expression. Then the cells were treated for 18h with the agonist, TNF-α; the conditioned medium was harvested and analyzed for MMP-2 and -9 levels.
10.4
REASEARCH ACHIEVEMENTS
10.4.1 Flow equipment
We set up two flow equipment: one for the oscillatory (fig.1a,b) and the other one for the laminar shear stress (fig. 2a,b). The apparatus we had made allowed us to run 4 samples of oscillatory and 4 samples of laminar at the same time. HUVECs were exposed for 24h to different flow conditions: static (0 dyne/cm2), oscillatory shear stress (±5 dyne/cm2), physiological laminar shear stress (15 dyne/cm2) and high laminar shear stress (45, 60, 75 dyne/cm2).
Fig.1b Oscillatory shear stress apparatus.
Fig.2b Laminar shear stress apparatus.
HUVECs exposed to laminar shear stress for 24h become elongated and aligned themselves to the flow direction. On the contrary, HUVECs exposed to oscillatory shear stress become apoptotic (Fig. 3).
Fig.3a HUVECs exposed to static, pathologic (±5 dyne/cm2) or physiologic (15 dyne/cm2) flow condition.
Oscillatory shear stress (±5 dyne/cm2)
No shear stress Laminar shear stress
10.4.2 Gelatin zymography
We found that the MMPs in serum and therefore in the media interfered with the determination of those secreted by the cells. To avoid this, we used a HUVEC line which needed only 2% serum; furthermore, the residual galatinases were stripped out of the media before treating the cells.
We found that the hydrocortisone contained into the media inhibited the MMPs production by TNF-α stimulated cells. To avoid this, we remove the hydrocortisone from the media before the treatment with TNF-α.
After 24h of flow experiment a big volume of conditioned media was obtained and therefore the gelatinases were too diluted to be detected. To avoid this, the conditioned media were concentrated many times by spin column centrifugation.
The same viscosity of plasma was obtained by adding 3.35% dextran to the media. Unfortunately, this conditioned media couldn’t be concentrate many times as the one without dextran.
HUVECs exposed to oscillatory shear stress showed an increased activity of MMP-2 and MMP-9 if compared with the one exposed to high laminar shear stress (fig. 4).
Fig. 4 High laminar shear stress versus oscillatory shear stress.
MMP-9
10.4.3 Real Time(RT)-PCR
HUVEC exposed to normal laminar shear stress (LSS) showed low expression of VCAM-1 which was increased by oscillatory shear stress (OSS) (100 fold) and moreover by static condition (1000 fold).
HUVEC exposed to normal laminar shear stress showed low expression of NOX-2 which was increased by static condition (3000 fold) and moreover by oscillatory shear stress (4000 fold).
0,001 0,010 0,100 1,000 10,000 100,000 STATIC OSS LSS 0,001 0,01 0,1 1 10 LSS OSS STATIC
HUVEC exposed to normal laminar shear stress (LSS 15) showed low expression of NOX-4 which was increased by oscillatory shear stress and by static condition (both 15 fold). Interestingly, high laminar shear stress 45 dyne/cm2 didn’t up-regulate NOX-4 which was increased by high laminar shear stress 60 dyne/cm2 (2 fold) and 75 dyne/cm2 (3 fold).
HUVEC exposed to normal laminar shear stress showed low expression of NOX-5 which was increased by oscillatory shear stress (2,5 fold) and moreover by static condition (90 fold). Interestingly, the expression of NOX-5 was slightly increased by high laminar shear stress 45 dyne/cm2 (1,5 fold) and moreover by 60 dyne/cm2 (120 fold) and 75 dyne/cm2 (45 fold).
1,0 10,0 100,0 1000,0 STATIC OSS LSS 15 LSS 45 LSS 60 LSS 75 1,0 10,0 100,0 STATIC OSS LSS 15 LSS 45 LSS 60 LSS 75
eNOS expression in HUVEC exposed to normal laminar shear stress was down-regulated by oscillatory shear stress (3 fold) and moreover by static condition (7 fold). Interestingly, high laminar shear stress 45 dyne/cm2 suppressed the expression of eNOS (25 fold).
HUVEC exposed to normal laminar shear stress showed high expression of HO-1 which was reduced by oscillatory shear stress (5 fold) and moreover by static condition (160 fold).
0,10 1,00 10,00 STATIC OSS LSS 15 LSS 45 0 1 10 100 1000 LSS OSS STATIC
HUVEC exposed to normal laminar shear stress showed high expression of NOX-1 which was suppressed by static condition (2500 fold) and moreover by oscillatory shear stress (500000 fold). Interestingly, high laminar shear stress 45 dyne/cm2 (500000 fold) and 60 dyne/cm2 (20000 fold) suppressed the expression of NOX-1.
KLF-2 expression in HUVEC exposed to normal laminar shear stress was down-regulated by static condition (10 fold) and moreover by oscillatory shear stress (35 fold).
1 10 102 103 104 105 106 107 108 109 STATIC OSS LSS 15 LSS 45 LSS 60 0,1 1,0 10,0 100,0 STATIC OSS LSS
10.4.4 Western Blot
We performed several western blots to assess the efficacy of virus infection but the primary antibody worked only the first time we used it and so we tested several antibodies until we discovered that also the virus was not working anymore.
10.4.5 HUVEC infection with the dominant negative (DN) PKC virus
To establish the viral titer required for 100% infection, HUVECs were infected with increasing multiplicity of infection (0, 100, 250, 500, 750 and 1000) of virus for 16 h. This was followed by an additional 24 h incubation in complete medium, to allow transgene expression. Viral infection efficiency was then assessed by histochemical staining for β-galactosidase (Fig. 5). To establish the best ratio of infection with DN-PKC and Tet-OFF viruses, HUVECs were infected with 3 different ratio: 1:1, 1:3, 1:9 (data not shown).
Fig. 5 Viral infection efficiency assessed by histochemical staining for β-galactosidase in HUVEC infected with
virus for 16h.
0 pfu 100 pfu 250 pfu
10.5 SUMMARY AND CONCLUSIONS
We aided setting up new flow equipment and established standard conditions to perform experiments and optimised protocols.
We overcame problems with PKC-ζ DN tet regulated overexpression vector; we have now obtained CMV-driven vector to perform further studies.
We found an increase in MMPs activity in HUVECs exposed to oscillatory shear stress and a decrease if exposed to high laminar shear stress.
We established a panel of control genes that are regulated by high shear stress for quality control of future samples.
VCAM-1, NOX-2, NOX-4 and NOX-5 expression was up-regulated in pathological condition such as oscillatory shear stress and in static condition if compared with normal laminar shear stress; VCAM-1, NOX-2, NOX-4 and NOX-5 behaved as pro-atherosclerotic genes in our system. In this regard, NOX-4 and NOX-5 expression were almost the same until high shear stress 45 dyne/cm2 and were up-regulated by very high laminar shear stress (60 and 75 dyne/cm2) reaching values of pathological condition.
eNOS, HO-1, NOX-1 and KLF-2 expression was down-regulated in pathological condition such as oscillatory shear stress and in static condition if compared with normal laminar shear stress; eNOS, HO-1, NOX-1 and KLF-2 behaved as atheroprotective genes in our system. In this regard, eNOS and NOX-1 were down-regulated by high laminar shear stress (45 and 60 dyne/cm2) reaching values of pathological condition.
High laminar shear stress 45 dyne/cm2 down-regulate the expression of eNOS and NOX-1 modulating atheroprotective genes whereas very high laminar shear stress 60 and 75 dyne/cm2 up-regulated the expression of NOX-4 and NOX-5 and down-regulated the expression of NOX-1 modulating both proatherosclerotic and atheroprotective genes in an opposite manner.
The following steps will be: to find other genes modulated by flow and to consolidate the preliminary data obtained until now; to see if the remodelling phenotype shown by HUVECs exposed to pathological flow conditions involves PKCζ and if its inhibitors can reverse it.
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