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LOW-INTENSITY SHEAR STRESS INCREASES ENDOTHELIAL ELR+ CXC CHEMOKINE PRODUCTION VIA A FAK-P38β MAPK-NF-κB PATHWAY

Sadiq S. Shaik1, Thomas D. Soltau1, Gaurav Chaturvedi2, Balagangadhar Totapally3, James S. Hagood1,4, William W. Andrews5, Mohammad Athar6, Nikolai N. Voitenok7, Cheryl Killingsworth8, Rakesh P. Patel9, Michael B. Fallon7,10, Akhil Maheshwari1,4,11

Departments of 1Pediatrics, 4Cell Biology, 5Obstetrics and Gynecology, 6Dermatology, 8Internal

Medicine, and 9Pathology, University of Alabama at Birmingham, Birmingham, Alabama; 11Translational Research in Normal and Disordered Development (TReNDD) Program, University of Alabama Department of Pediatrics; 7Fund for Molecular Hematology &

Immunology, Moscow, Russia;7Veterans’ Affairs Hospital, Birmingham, Alabama; 2Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas; and

3Pediatrics, Miami Children's Hospital, Florida Address correspondence to: Akhil Maheshwari, MD, NHB 525, 619 19th St South, Birmingham, AL 35233. Phone: 205-934-4680; Fax: 205-212-2014; E-mail: akhil@peds.uab.edu

CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR+ CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well-elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR+ CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR+ CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low-intensity shear stress. Low shear activated endothelial ELR+ CXC chemokine production via cell surface heparan sulfates, β3-integrins, focal adhesion kinase (FAK), p38β mitogen-activated protein kinase (MAPK), mitogen and stress-associated protein kinase (MSK)-1, and nuclear factor (NF)-κB.

The CXC family of chemokines is defined by a conserved ‘C-X-C’ sequence near the N-terminus, where two cysteine (C) residues are separated by a non-conserved amino acid (X) (1,2). These chemokines are further sub-classified on the basis of the presence or absence of an additional glutamate-leucine-arginine (ELR) motif into ELR+ CXC and the ELR– CXC groups (1). The vascular endothelium is an important source of ELR+ CXC chemokines, where these chemokines serve as important chemoattractants for neutrophils and monocytes and also play an important role in angiogenesis (3-6). There are seven known ELR+ CXC chemokines in humans, including interleukin-8 (IL-8)/CXC ligand 8 (CXCL8), growth related oncoprotein (GRO)-α/CXCL1, GRO-β/CXCL2, GRO-γ/CXCL3, epithelial-derived neutrophil attractant (ENA)-78/CXCL5, granulocyte chemotactic peptide (GCP)-2/CXCL6, and the neutrophil-activating peptide (NAP)-2/CXCL7 (2).

ELR+ CXC chemokines are critical regulators of leukocyte trafficking into inflamed tissues (4,5,7,10,11). For reasons that are not well-elucidated, endothelial-leukocyte interaction and leukocyte emigration into the tissues is primarily concentrated in small vessels such as capillaries and post-capillary venules (PCVs) (8). This microvascular localization of endothelial-leukocyte interaction is only

http://www.jbc.org/cgi/doi/10.1074/jbc.M807205200The latest version is at JBC Papers in Press. Published on December 31, 2008 as Manuscript M807205200

Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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partially explained by the lower intensity of the hemodynamic shear forces in smaller vessels, which, in terms of possible mechanical interference, may be more conducive to leukocyte binding to the vessel wall than the higher shear intensities seen in the arterial trunks (7,12). The magnitude of shear stress in capillaries and post-capillary venules is ≤5 dynes/cm2 (≤0.5 pascal), which is significantly lower than the 10-30 dynes/cm2 (1-3 pascals) in arteries (13). Because ELR+ CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Hemodynamic shear is an important modulator of gene expression in endothelial cells (14-17), but there are conflicting data on the effect of shear stress on IL-8/CXCL8 expression in endothelial cells (14,15,17,18). Therefore, we further investigated whether shear-induced chemokine expression in endothelial cells is related to the intensity of shear stress.

Using both in vitro and in vivo models, we show that ELR+ CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells, and that these differences can be explained on the basis of the preferential activation of endothelial chemokine production by low-intensity shear stress. Low shear activation of endothelial ELR+ CXC chemokine production involves a signaling pathway comprised of cell surface heparan sulfates, β3-integrins, focal adhesion kinase (FAK), p38β mitogen-activated protein kinase (MAPK), mitogen and stress-associated protein kinase (MSK)-1, and nuclear factor (NF)-κB.

Experimental Procedures:

Animals: All animal studies were approved by the local Institutional Animal Care and Use Committee. Three mixed-breed pigs (10 ± 4 kg) were anesthetized by intramuscular telazol (4.4 mg/kg), xylazine (4.4 mg/kg), atropine (0.04 mg/kg), and inhaled isoflurane, and were mechanically ventilated in a dorsally recumbent position. Right carotid artery and left jugular vein were cannulated and blood samples were

drawn from the cannulae. Capillary blood samples were collected using a glass capillary tube from a 1 inch long, 3 mm deep incision on the ventral surface of the right ear. Tissues (aorta, heart, jejunum, and kidney) were harvested after euthanasia. Endothelial cells: Human umbilical venous endothelial cells (HUVEC) were isolated by trypsin treatment of umbilical cord veins (19). Umbilical cords and cord blood were obtained from term placentae after approval by the local Institutional Review Board. Cells were seeded on flasks coated with a mixture of fibronectin (10 µg/mL; Sigma, St. Louis, MO) and gelatin (2% w/v, Sigma) and grown in the complete EGM-2 medium (Lonza Biosciences, Walkersville, MD). Cultures were purified by positive immunoselection using anti-CD31 ferromagnetic microbeads (Miltenyi, Auburn, CA) as per manufacturer's instructions. Experiments were performed on culture passages 3-4. HAEC and HMEC (pulmonary, dermal) endothelial cells (Lonza) were used at passages 3-5. Shear stress: Endothelial cells were grown to confluence in 100 × 20 or 35 × 10 mm dishes and exposed to shear stress by using a cone-plate viscometer as described elsewhere (20). The cone-plate system is one of two devices commonly used to apply shear stress in vitro, the other being a parallel-plate flow chamber (PPFC) (21). The cone-plate system provides a wider range of shear intensities and in greater uniformity of shear forces throughout the field as compared to the PPFC (21,22). Using a static viscosity = 0.8 centipoise for culture media (20), the shear stress applied with our cone at 6 rotations/sec was computed as 20 dynes/cm2. Shear intensities of 4 and 10 dynes/cm2 were attained by variation of rotational speed. We used 20 dynes/cm2 to represent the wall shear stress in larger arteries (23), 4 dynes/cm2 to simulate the physical conditions in capillaries and venules (24), and an intermediate intensity (10 dynes/cm2). Cell viability (trypan blue exclusion) was unchanged after shear. Neutrophil chemotaxis: Neutrophil chemotaxis was measured in microchemotaxis chambers as described previously (details provided in the online supplement) (5). We used supernatants from endothelial cultures in lower wells of

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microchemotaxis chambers (NeuroProbe, Gaithersburg, MD). The number of migrating cells was read off a standard curve generated from known numbers of labeled cells. Real-time PCR: Total RNA was isolated using the Trizol reagent (Ambion) as per the manufacturer’s instructions. Primers were designed using Beacon Design software (Bio-Rad, Hercules, CA). Real time PCR protocol and the primer sequences are described in the online supplement (25). Data were normalized for GAPDH and gene expression was compared between samples by using the 2–ΔΔCT method. Measurement of chemokines: Human, porcine, and rat chemokines were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits; porcine IL-8, rat CINC-1/CXCL1, human IL-8/CXCL8, GCP-2/CXCL6, GRO-a/CXCL1, ENA-78/CXCL2, NAP-2/CXCL7 from R&D, Minneapolis, MN; GRO-β/CXCL2 from Immunobiological labs, Hamburg, Germany; and GRO-γ/CXCL3 from Antigenix, Huntington Station, NY). Finally, to determine the effect of low vs. high shear on the cytokine/chemokine expression profile of endothelial cells, we used a real-time PCR microarray to measure mRNA expression of 89 major chemokines and cytokines (SA Biosciences, Frederick, MD). Reagents: Polyclonal anti-FAK, phospho-FAK (ser722), p38 and phospho-p38, MSK1, and NF-κB p65 and phospho-NF-κB p65 (ser276) were from Santa Cruz Biotechnology. Monoclonal rabbit anti-phospho-MSK1 (ser376), polyclonal anti-phospho-integrin β1 (ser785) and integrin β3 (tyr773), and neutralizing rabbit anti-integrin β3 antibodies were from Chemicon-Millipore. We used inhibitors of p38 kinase (SB202190), MSK-1 (Ro318220), NF-κB (N-Tosyl-l-phenylalanine chloromethyl ketone (TPCK), MG-132, pyrrolidine dithiocarbamate (PDTC), parthenolide, and curcumin), PI3K (wortmannin), and rho kinase (Y-27632), Flavobacterium heparinase and TNF-α, all from Sigma. Immunocytochemistry: HUVEC grown on glass coverslips were exposed to shear for ≤6 hrs. The immunocytochemistry protocol is described in the online supplement (26).

Western blots and mitogen-activated protein kinase (MAPK) antibody array analysis: Western blots were performed using standard protocols and were analyzed using the NIH scion software (27). The MAPK array (R&D) is designed to analyze extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK1-3), p38 α/β/γ/δ, Akt1-3, GSK-3α/β, and p70 S6 kinase. Electrophoretic mobility shift assay (EMSA): The EMSA protocol has been described previously (28). We used the consensus binding sequence (underlined) for NF-κB (5'-AGTTGAGGGGACTTTCCCAGG C-3' pre-labeled with an infrared fluorophore (Licor, Lincoln, NE). For supershift assay, the nuclear extracts were incubated with the anti-p65

antibody (1 µg, 30 min on ice). Gels were analyzed in an infrared scanner (Licor). Plasmids and RNA interference: pCMV-Neo-FAK and pCMV-Neo-FRNK plasmids (Dr. Tom Parsons, University of Virginia),(29) the ΔNF-κB 162/+44 hIL-8/Luc plasmid, generated by site-directed mutagenesis of the NF-κB site in

the context of the -162/+44 hIL-8 (Dr. Allan Brasier, University of Texas) (30) and pcDNA3-FLAG plasmids expressing the wild-type p38 isoforms (Dr. Jiahuai Han, The Scripps Research Institute) (31) have been described previously. We used a mammalian p38β2 small interfering RNA (siRNA) expression plasmid (pKD-p38β/SAPK2b2-v1, Upstate) to knock down p38β2 and used the cells after 48 hrs. p38δ was knocked down by using siRNA oligonucleotides (5'-GGACCAGUAUUUGUCACUAtt; Ambion, Austin TX) and the cells were used after 24 hrs. MSK-1 siRNA oligos are commercially available (Santa Cruz Biotechnology). Negative

controls were maintained with plasmid backbone or control siRNA (Ambion). HUVEC were transfected with plasmid DNA (1 µg) or oligos (10 nM, pre-determined optimum) using FuGENE 6 (Roche, Indianapolis, IN) as per the manufacturer’s protocol. Transfection efficiency was measured in controls transfected for GFP (pMaxGFP, Amaxa, Gaithersburg, MD). Luciferase assays: Cell extracts after 8 hrs of shear stress were used in the Luciferase Assay System (Promega) as per manufacturer’s instructions.

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Statistical methods: Non-parametric tests were applied using the SigmaStat 3.1.1 software (Systat, Point Richmond, CA). A p value of <0.05 was considered significant.

Results:

ELR+ CXC chemokine production by microvascular and aortic endothelial cells: To determine whether ELR+ CXC chemokines are differentially expressed by microvascular and aortic endothelium, we first compared IL-8/CXCL8 and GRO-α/CXCL1 production by low-passage human microvascular endothelial cells (HMEC) and human aortic endothelial cells (HAEC) in vitro. As seen in Fig. 1A, basal chemokine production in HMEC was higher than HAEC. HMEC chemokine production was also higher than HAEC following stimulation with 1-10 ng/mL TNF-α or E. coli lipopolysaccharide (10-500 ng/mL; data not depicted). These data were supported by subsequent in vivo measurements of porcine IL-8 in simultaneously drawn arterial, venous, and capillary blood samples from anesthetized pigs. Porcine IL-8 concentrations in capillary/venous blood were higher than in arterial samples (Fig. 1B), providing further indirect evidence for a microvascular origin for circulating ELR+ CXC chemokines. There was also strong immunoreactivity for IL-8 in microvascular but not in aortic endothelial cells (inset). Low-intensity laminar flow shear increases ELR+ CXC chemokine expression in endothelial cells: To determine the mechanism(s) underlying the observed differences in chemokine expression by microvascular and aortic endothelium, we next investigated the hypothesis that low-intensity shear stress in capillaries and veins (24) is a more efficient activator of chemokine expression in vascular endothelial cells than the higher shear intensities seen in arteries. We exposed HAEC, HMEC, and human umbilical venous endothelial cells (HUVEC) to low (3 dynes/cm2) and high (20 dynes/cm2) laminar flow shear stress and measured IL-8/CXCL8 and GRO-α/CXCL1 production over 18 hrs. Low-intensity shear increased chemokine production in HAEC and HMEC, whereas high shear suppressed chemokine expression to below static levels

(Fig. 2A). To confirm the functional relevance of the chemokine levels we measured in our culture supernatants, we next measured the ability of these supernatants to recruit neutrophils in vitro. As seen in Fig. 2B, low, but not high, shear increased the neutrophil chemotactic activity in endothelial culture supernatants.

The differential effects of various shear intensities on endothelial ELR+ CXC chemokine expression were similar in HUVEC, which justified the use of HUVEC to model endothelial cells in subsequent experiments. The effect of low, intermediate (10 dynes/cm2), and high-intensity shear on the four highest upregulated ELR+ CXC chemokines (IL-8/CXCL8, GRO-α/CXCL1, GRO-β/CXCL2, and GRO-γ/CXCL3), which are also the most potent neutrophil chemoattractants in this group (5), is summarized in Fig. 2C. As shown in supplementary fig. 1, low shear activates the IL-8/CXCL8 gene promoter, which provides a mechanism for the observed increase in mRNA/protein expression. Besides the four aforementioned chemokines, low shear also upregulated GCP-2/CXCL6, ENA-78/CXCL5, and NAP-2/CXCL7. Low shear increased GCP2/CXCL6 levels from undetectable, 251±8, and 253±11 pg/mL at 8, 16, and 24 hrs of static culture, respectively, to 22 ± 5, 322±3, and 332±50 pg/mL (p<0.01), In contrast, levels decreased with intermediate shear to undetectable, 32±11, and 32±3 pg/mL and to undetectable, 36±11, and 32±13 pg/mL, respectively with high shear (p<0.05 compared to static controls). Low shear also increased ENA-78/CXCL5 concentrations but both static and shear-treated levels were <25 pg/mL, levels that did not produce measurable neutrophil chemotactic activity in vitro (5). NAP-2/CXCL7 concentrations in static cultures were uniformly close to the lower limit of detection but increased to 34±3 pg/mL and 187±21 pg/mL after 16 and 24 hrs of low shear, respectively (p<0.05). Intermediate and high shear downregulated all time points to <25 pg/mL (p<0.05).

We also tested the cell lysates to measure intracellular and membrane-bound chemokines, which play an important role in endothelial-neutrophil interaction (32). Similar to changes in secreted chemokines, low shear upregulated IL-

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8/CXCL8 and GRO-α/CXCL1 concentrations, whereas higher shear intensities suppressed the expression of these two chemokines. Shear did not change GRO-β/CXCL2, GRO-γ/CXCL3, and NAP-2/CXCL7, whereas GCP-2/CXCL6 remained low near the lower limit of detection. We next used a real-time PCR microarray to investigate the effect of low and high shear intensities on the global cytokine/chemokine expression profile of endothelial cells. As seen in Fig. 2B, low shear increased the expression of a large number of chemokines and cytokines. In contrast, high shear suppressed the expression of all chemokines (including the CXC, CC, C, and the CX3C families), but differentially altered the expression of other cytokine subgroups. These data are consistent with our hypothesis that low-intensity shear stress increases chemokine expression in microvascular endothelium, which may explain, at least partially, the microvascular localization of endothelial-leukocyte interaction. Cell surface heparan sulfates, β3-integrins, and FAK transduce the mechanical signal of low-intensity shear stress: To determine the signaling mechanism by which low shear upregulates chemokine expression, we first investigated whether disruption of cell surface heparan sulfate proteoglycans (HSPGs), which are important endothelial mechanotransducers (33,34), can block low shear-induced chemokine production. We used a previously reported protocol of heparinase treatment (15 units/mL x 2 hrs) that has been shown to remove a substantial fraction of the HSPGs. As shown in Fig. 3A, heparinase treatment blocked low shear-induced IL-8 and GRO-α production.

Endothelial surface HSPGs bind to fibronectin and collaborate with integrins to recruit phosphorylated FAK (33,34). To identify the involved integrins, we used a PCR array to short-list fibronectin-binding integrins that are ‘activated’ by shear. Low shear increased the transcription of α3, αv, β1, and β3 integrin genes (supplementary fig. 2). The activation of β1- and β3- integrins was further investigated by immunocytochemistry. After exposure to low shear for 4 hrs, 93±4% cells stained positive for phospho-β3-integrins (Fig. 3B) whereas phospho-β1 was detected in 31±3% cells. Neutralization of phospho-β3-integrins partially

blocked low shear-induced chemokine production (inset).

We next investigated the role of FAK in low shear-induced chemokine production. As shown by western blot and immunocytochemistry in Fig. 3C, low shear phosphorylated FAK in endothelial cells. Upregulation of FAK by transient transfection increased low shear-induced chemokine production, whereas inhibition of FAK expression by over-expressing FRNK (FAK-related non-kinase) blocked chemokine production. Shear-induced ELR+ CXC chemokine expression requires p38β and MSK-1: An antibody array identified p38β and p38δ as the two activated MAPKs in low shear-exposed cells (supplementary fig. 3). Shear-induced p38 phosphorylation was confirmed by western blotting and immunocytochemistry (Fig. 4A). However, low-shear activation of endothelial chemokine production was completely blocked by SB202190, which inhibits p38β, but not p38δ (Fig. 4B). Furthermore, when individual p38 isoforms were over-expressed in endothelial cells through transient transfection, p38β was the most efficient p38 isoform in upregulating shear-activated chemokine expression (Fig. 4C). We further confirmed these data by using RNA interference to knock-down p38β2 (the major splice variant of p38β)(35) and p38δ; downregulation of p38β2, but not p38δ, blocked low shear-induced chemokine production (Fig. 4D).

Among the downstream targets of p38, shear is a known activator of MSK-1 and the ribosomal S6 kinase (RSK)-2 (36). MSK-1 activation in endothelial cells is almost exclusively dependent on p38 signaling (36) and therefore, we investigated the role of MSK-1 in shear-induced chemokine production. As shown in Fig. 5A, low shear phosphorylated MSK-1. Inhibition of MSK-1 activity (Ro318220) partially blocked chemokine production (Fig. 5B), indicating that MSK-1 is an important, although possibly not the sole mediator of shear-induced chemokine production. Low shear-induced ELR+ CXC chemokine production does not require rho-associated protein kinase (ROCK) or phosphatidylinositol 3-kinase (PI3K): ROCK and PI3K induce endothelial chemokine expression during

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inflammation (37). However, inhibition of these enzymes by using wortmannin and Y-27632, respectively, did not block low shear-induced chemokine production in our system (not depicted). Shear-induced ELR+ CXC chemokine expression occurs via NF-κB activation: We next investigated the role of NF-κB, which is an important transcriptional regulator of ELR+ CXC chemokines and is also a major downstream target of MSK-1 (38). We first sought ‘shear-activated’ NF-κB genes in a PCR-based array. Low shear increased the transcription of NFKB2 (p52/p100), REL, and REL A/p65 (supplementary fig. 4). Because REL A/p65, the most highly upregulated gene in this family following exposure to low-intensity shear, is a well-known substrate for MSK-1, we used p65 in subsequent experiments to shear activation of the NF-κB pathway.

Low shear caused the phosphorylation of NF-κB p65 ser276 (Fig. 6A), increased specific binding of NF-κB proteins to DNA (Fig. 6B), and NF-κB-mediated activation of IL-8 promoter (Fig. 6C). We next used signaling inhibitors to block various steps of the NF-κB pathway (39): (a) inhibitors of I-κB degradation including N-Tosyl-l-phenylalanine chloromethyl ketone (TPCK, a serine/cysteine proteases inhibitor) and MG-132 (proteasomal inhibitor); (b) pyrrolidine dithiocarbamate (PDTC, inhibits nuclear translocation of NF-κB proteins); (c) parthenolide (inhibits I-κB kinases); and (d) curcumin, which prevents NF-κB activation upstream of the I-κB kinases. However, all inhibitors except curcumin induced apoptotic changes in endothelial cells as early as 5-6 hrs. Curcumin inhibited low shear-induced chemokine mRNA and protein expression (supplementary fig. 5) and established the quantitation of mRNA as an early measure of chemokine production. TPCK, MG-132, PDTC, and parthenolide blocked shear-induced chemokine mRNA expression at 4 hrs, an early time-point selected before the onset of apoptotic changes associated with NF-κB inhibitors (Fig. 6D). Shear-induced intracellular signaling is comprised of discrete, sequential steps: To confirm the sequence of activation of FAK, p38, MSK-1, and NF-κB, the major signaling

mediators involved in low shear activation of chemokine production, we repeated western blots to measure phosphorylation in the presence of specific inhibitors. As shown in Fig. 7A, downregulation of FAK activity by FRNK inhibited the phosphorylation of p38, MSK-1, as well as NF-κB p65. In contrast, p38 inhibition did not affect FAK, but prevented the activation of MSK-1 and NF-κB. Finally, inhibiting MSK-1 activity did not affect the phosphorylation of FAK and p38 but blocked the phosphorylation of p65. To ensure that these results were not affected by possible non-specific effects of the inhibitors on other signaling mediators, we further confirmed these data by using specific siRNA to knockdown p38β and MSK-1 (supplementary fig. 6). The signaling pathway mediating low shear-induced chemokine expression is summarized in Fig. 7B. Discussion:

We present a detailed analysis of the signaling mechanisms by which low-intensity shear stress activates ELR+ CXC chemokine production in endothelial cells. Differences in the effects of low vs. higher shear intensities can explain the differential expression of chemokines we observed in microvascular and aortic endothelial cultures. Our findings are in agreement with previous reports of Chen et al.(15) and Cheng and colleagues (17) but contrast with the observations of Kato et al.(18) and Partridge and coworkers (40), who reported that shear stress suppressed endothelial IL-8 production. Kato (18) and Partridge (40) used relatively high shear intensities of 7 and 12 dynes/cm2, respectively, which can explain the observed differences.

We have shown that low-intensity shear stress activates endothelial ELR+ CXC chemokine production via a HSPG/β3-integrin/FAK/p38β/MSK-1/NF-κB pathway. The important role we observed for endothelial HSPGs is consistent with previous reports of HSPG effects in mechanotransduction (33,34). Endothelial glycocalyx is the thickest in the microvasculature (41), the site of chemokine production in our studies, and consistent with our data that heparan sulfates (present in the glycocalyx) are involved in chemokine expression. Furthermore, HSPGs can amplify

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the effects of intra-circulatory chemokine gradients by immobilizing the chemokines to the endothelial cell membrane, thereby facilitating detection by migrating leukocytes. Using immuno-electron microscopy, Middleton and colleagues demonstrated that endothelial cells in postcapillary venules and small veins, but not in other blood vessels, display IL-8/CXCL8 on the luminal surface (42). HSPGs interact with fibronectin-binding integrins and FAK (33,34), and consistent with these reports, we detected that low shear activated β3- and β1-integrins. Although we did not investigate individual integrins, there is evidence that αvβ1, αvβ3, α3β1, and α3β3 integrins identified in our PCR array are involved in focal adhesions and intercellular attachments (43,44).

Similar to our findings, FAK-induced p38 activation has been observed in cardiac myocytes, where mechanical stretch phosphorylates FAK to generate a binding site for grb2, a scaffolding protein that links FAK to the MAPK pathway (45). In endothelial cells, Li et al.(46) reported a transient activation of p38 after exposure to 12 dynes/cm2 shear but they did not measure the effect of lower intensities. Although the role of p38 in shear-induced ELR+ CXC chemokine expression has not been examined earlier, p38 is a known regulator of endothelial chemokine expression in response to environmental stress, ultraviolet light, heat, osmotic shock, microbial products, and inflammatory cytokines (47). We also demonstrate the importance of the p38β isoform in shear effects, which is consistent with previous observations that endothelial cells express p38β as a major isoform and are relatively deficient in p38δ (35). The activation of p38β is interesting because unlike p38α, p38β has an anti-apoptotic effect in endothelial cells, which is similar to the cytoprotective effects observed with both shear stress as well as ELR+ CXC chemokines (48,49). Further studies are needed to determine whether anti-apoptotic effects of shear in endothelial cells are mediated via ELR+ CXC chemokines.

In our study, p38 activated MSK-1, which, in turn, phosphorylated NF-κB p65. MSK-1 is a key nuclear regulator of NF-κB p65-mediated transcription (50). The partial inhibition of low shear-mediated chemokine expression by

Ro318220, which inhibits MSK-1, can be explained because other kinases including casein Kinase II, Akt, I-κB kinases, protein kinase A and the p38 MAPKs can also phosphorylate p65 (38). p38 may also increase NF-κB-mediated gene transcription by increasing I-κB degradation and thereby promoting the translocation of the translocation of NF-κB proteins into the nucleus (51,52). The involvement of the NF-κB pathway in shear activation of endothelial chemokine production is consistent with previous reports of shear augmentation of the effects of inflammatory agents such as reactive oxygen species and TNF-α on NF-κB activation (40,53,54).

The preferential activation of endothelial chemokine expression by low shear is consistent with the concentration of endothelial-leukocyte interaction in capillaries and PCVs. Further work is needed to investigate the role of endothelial-derived ELR+ CXC chemokines in the normal non-inflammatory circulation of leukocytes. These chemokines may provide a mechanism for the high neutrophil concentrations seen in the capillaries in the physiological state. During flow through narrow blood vessels, blood cells are normally displaced towards the axis of the vessel to create a cell-depleted wall region, which facilitates flow by lowering the apparent viscosity (the Fahraeus-Lindqvist effect) (55). At branch points, this cell-depleted wall region allows a progressively smaller fraction of cells to enter the daughter vessel than the parent vessel, causing a progressive reduction in hematocrit as blood courses its way from arterioles to capillaries (55). However, in contrast to RBCs, the neutrophil concentrations in capillaries are up to 40-80 times higher than in larger blood vessels (56,57). Although some differences can be explained by the longer neutrophil transit times through capillaries, chemokine gradients may promote neutrophil circulation via narrow capillaries instead of the larger ‘thoroughfare’ arteriolovenular shunts (58). Sources of Funding: Supported by the American Heart Association (AHA) grant 0665155B, American Gastroenterological Association Research Scholar Award, and NIH/NICHD K12HD043397 (A.M.), AHA award 0655312B (R.P.P), and NIH awards

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HL082818 (J.S.H) and DK056804 (M.B.F). The work was made possible in part by Research Facilities Improvement Program Grant C06 RR 15490 from the National Center for Research Resources. Disclosures: The authors have no conflicts to report. Author contributions: S.S.S., T.D.S., G.C. performed experiments; B.T., J.S.H., W.W.A., M.A., N.N.V., C.K., R.P.P., M.B.F. assisted in research design, provided vital reagents; A.M. designed the study, performed some experiments, and wrote the paper.

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Figure legends: Fig.1. Differential expression of ELR+ CXC chemokines by microvascular and aortic endothelial cells: A. Human microvascular

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endothelial cells produce more IL-8/CXCL8 and GRO-α/CXCL1 than aortic endothelium in vitro. Bar diagrams show mean concentrations (± standard errors) measured by ELISA. Groups compared by the Mann-Whitney U test; * denotes p<0.05. These data are representative of three separate experiments. B. Porcine IL-8 levels in tissue capillaries and veins are higher than in arterial blood. Bar diagrams show chemokine concentrations (means ± standard errors) measured by ELISA. Groups compared by the Kruskal-Wallis H test/Dunn's multiple-comparison post-test; * denotes p<0.05, N.S. indicates differences not significant. Inset: Microvascular, but not aortic, endothelium shows immunoreactivity for IL-8. Porcine tissues were stained with a monoclonal anti-porcine IL-8 antibody and appropriate secondary reagents. Open arrow shows the absence of IL-8 immunoreactivity in the aortic endothelium. A few smooth muscle cells stain positive for IL-8 in the tunica media. Solid arrows indicate IL-8 immunoreactivity in the microvessels in the heart, intestine, and kidney. These data represent studies on three animals. Fig.2A. Low-intensity shear stress increases endothelial ELR+ CXC chemokine production: a. Low, but not high, shear increased chemokine production above static levels in human aortic and microvascular endothelium. Bar diagrams show IL-8/CXCL8 and GRO-α/CXCL1 concentrations (mean ± standard errors) measured by ELISA. Groups compared by the Kruskal-Wallis H test/Dunn's multiple-comparison post-test; * denotes p<0.05, N.S. indicates differences not significant. These data are representative of three separate experiments, where each study group was in triplicate. b. Consistent with the chemokine concentrations, low, but not intermediate/high, shear increased neutrophil chemotactic activity in endothelial culture supernatants. Bar diagrams show mean number of migrating neutrophils (± standard errors) in a fluorescence-based microchemotaxis assay. We used culture supernatants from the microvascular endothelial culture shown in panel A. Groups were compared by the Kruskal-Wallis H test/Dunn's multiple-comparison post-test; * denotes p<0.05, N.S. indicates differences not

significant. The effect of low shear was blocked by excess monoclonal anti-CXCR2 antibody (not depicted). These data are representative of three separate experiments, where each study group was in triplicate. c. Low, but not intermediate/high, shear stress increased chemokine production in human umbilical venous endothelial cells (HUVEC). Panels show the effect of low, intermediate, and high shear intensities on the expression of IL-8/CXCL8, GRO-α/CXCL1, GRO-β/CXCL2, and GRO-γ/CXCL3, the four most highly upregulated ELR+ CXC chemokines. Line diagrams show serial chemokine concentrations (mean ± standard errors) in culture supernatants, compared by repeated measures analysis of variance on ranks; * denotes p<0.05, N.S. indicates differences not significant. Insets in each panel show serial measurements of chemokine mRNA (fold changes in chemokine mRNA above GAPDH as a function of time), and immunocytochemistry to show intracellular chemokines. These data showed that the results in HUVEC were similar to other endothelial types and justified the use of HUVEC as an endothelial model in subsequent experiments. These data are representative of at least five separate experiments. Fig.2B. Low and high shear intensities have an opposite effect on endothelial chemokine expression: a. Low-intensity shear stress differentially activated mRNA expression of several chemokines and cytokines in HUVEC at 8 hrs. Bar diagram (means ± standard errors) shows fold change in mRNA concentrations above static culture conditions. Broken horizontal line separates chemokines (subfamilies CXC, CC, C, and CX3C) from other major cytokines. A 2-fold change (solid vertical line) was accepted as a ‘major’ change with acceptable discriminatory value. b. In contrast to low shear, high shear suppressed the expression of chemokines but had a variable effect on other major cytokines. Bar diagram (means ± standard errors) shows fold change in mRNA concentrations at 8 hrs above static culture conditions. Data in these figures represent five separate experiments.

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Fig.3. Cell surface heparan sulfates, β3-integrins, and FAK are involved in low shear-induced chemokine production: A. Low shear-activated endothelial production of IL-8/CXCL8 and GRO-α/CXCL1 was abrogated by prior heparinase treatment. Line diagrams show serial chemokine concentrations (mean ± standard errors) in culture supernatants. Groups were compared by the repeated measures analysis of variance on ranks; * denotes p<0.05. B. Immunoreactivity for phospho-β3-integrins. Fluorescence photomicrographs show HUVEC on coverslips (400x); Phospho-β3-integrins are localized as green punctate spots along cellular margin. Higher magnification image (1000x) on the right shows green immunoreactivity on focal adhesions. Inset: Neutralization of phospho-β3 integrins partially inhibited low shear-induced chemokine production. Bar diagrams show IL-8/CXCL8 and GRO-α/CXCL1 concentrations (mean ± standard errors) measured by ELISA. Groups compared by Kruskal-Wallis H test/Dunn's post-test; * denotes p<0.05. These data are representative of three separate experiments, where each study group was in triplicate. C. Low shear-induced endothelial chemokine production is mediated through the focal adhesion kinase (FAK). A representative western blot shows shear-induced phosphorylation of FAK; bar diagram (means ± standard errors) shows average densitometric analysis of the blots. Measurements were compared by the Kruskal-Wallis H test/Dunn's multiple-comparison post-test; * denotes p<0.05. Inset: immunolocalization of phospho-FAK (arrows indicate focal adhesions). Bar diagram on the right shows that over-expression of FAK by transient transfection sensitized endothelial cells to low shear and increased chemokine production whereas downregulation of FAK by transfecting FRNK inhibited chemokine production. Bars show chemokine concentrations (mean ± standard errors) as measured by ELISA. Groups compared by the Kruskal-Wallis H test/Dunn's post-test; * denotes p<0.05. Inset: shows western blots showing FAK expression in wild-type and transfected endothelial cells used in these experiments. These data are representative

of three separate experiments, where each study group was in triplicate. Fig.4. Low shear increases endothelial chemokine production by activating p38β: A. A representative western blot shows shear-induced phosphorylation of p38 MAPK; bar diagram (means ± standard errors) shows average densitometric analysis of the blots; groups were compared by the Kruskal-Wallis H test/Dunn's post-test; * denotes p<0.05. Inset: immunolocalization of phospho-p38. B. Addition of SB202190, an inhibitor of p38α and p38β, inhibited low shear-mediated endothelial chemokine production in a dose-dependent fashion. Bars show chemokine concentrations (mean ± standard errors). Statistical methods as above. C. Bar diagram shows the effect of over-expression of p38 isoforms by transient transfection on low shear-induced endothelial chemokine production. Over-expression of p38β increased shear-induced endothelial chemokine production. Statistical methods as above. Transfection was confirmed by western blotting for a FLAG sequence in the plasmid. D. Knocking down p38β, but not p38δ, by RNA interference inhibited low shear-mediated endothelial chemokine production. Bars show chemokine concentrations (mean ± standard errors) as measured by ELISA. Statistical methods as above. Insets show western blots to confirm that the siRNA reagents effectively downregulated the expression of the specific isoform. Data in these figures are representative of at least three separate experiments, where each study group was in triplicate. Fig.5. Low shear activates MSK-1: A. A representative western blot shows shear-induced phosphorylation of MSK-1; bar diagram (means ± standard errors) shows average densitometric analysis of the blots. Measurements were compared by the Kruskal-Wallis H test/Dunn's post-test; * denotes p<0.05. Inset: immunolocalization of phospho-MSK-1. B. MSK-1 inhibition by adding Ro318220 partially inhibited low shear-mediated endothelial chemokine production. Bars show chemokine concentrations (mean ± standard errors) as measured by ELISA. Statistical

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methods as above. These data are representative of three separate experiments. Fig.6. Low shear activates the NF-κB pathway: A. A representative western blot shows shear-induced phosphorylation of NF-κB p65/rel A; bar diagram (means ± standard errors) shows average densitometric analysis of the blots. Measurements were compared by the Kruskal-Wallis H test/Dunn's post-test; * denotes p<0.05. Inset: Immunocytochemistry shows nuclear translocation and phosphorylation of NF-κB p65/rel A. B. Low shear increased specific p65 binding to the DNA, as detected in electrophoretic mobility shift assay. Lanes 1-3 represent nuclear extracts from static cultures, whereas lanes 4-6 show extracts after exposure to low shear x 2 hrs. Each set includes the incubation of the nuclear extract with a probe specific for NF-κB, a mutated (scrambled) probe, and with the specific probe and a specific antibody (supershift). C. Low shear-activated IL-8/CXCL8 transcription is mediated via NF-κB, as seen in luciferase reporter assays utilizing constructs with the wild type and NF-κB site-mutated IL-8 promoter sequence. Bar diagram (means ± standard errors) shows relative light unit measurements; statistical analysis was as above. D. Low shear-induced chemokine mRNA expression was blocked by NF-κB inhibitors including agents that inhibit I-κB kinases (parthenolide), block I-κB degradation (N-Tosyl-l-phenylalanine chloromethyl ketone (TPCK) and MG-132), and inhibit the nuclear translocation of NF-κB proteins (pyrrolidine dithiocarbamate, PDTC). These agents were used for 4 hrs because of onset of apoptotic changes in endothelial cells with longer exposures. Bar diagram shows fold changes in chemokine mRNA above GAPDH (means ± standard errors) as a function of time; statistical analysis as above. Data in these figures represent at least three separate experiments. Fig.7. Low shear-induced endothelial signaling involves a series of discrete, sequential steps: A. Inhibition of FAK by transient transfection with FRNK blocked low shear activation of p38, MSK-1, and NF-κB. p38 inhibition blocked MSK-1 and NF-κB activation, but not that of FAK. Similarly, MSK-1 inhibition did not affect

FAK and p38 but prevented NF-κB activation. B. Proposed signaling pathway involved in low shear-mediated endothelial chemokine production. p38 MAPK is shown at the cusp of cytoplasm and nucleus because the site of its activation is not exactly known. These data are representative of at least three separate experiments.

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(C) ACTIVATION OF IL-8 PROMOTER (D) NF-κB INHIBITORS

Relative Light Units

0

100

80

60

40

20

1

2

3

0 250 500 750 1000

Static WT IL-8 promoter

Low shear WT IL-8 promoter

Low shear Mutant IL-8 promoter

(ΔNF-κB)

(A) PHOSPHORYLATION OF NF-κB (B) NF-κB BINDING TO DNA

*

**

**

*

****

**** **** **** **** **** ****

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Static Low Shear

Low shear

+ FAK

Low shear

+ FRNK

Static Low Shear

Low shear +

SB202190

Static Low Shear

Low shear +

Ro318220

β3-integrins

Heparan sulfate proteoglycans

Laminar flow shear

FAK

p38β SAPK

NUCLEUS

CYTOSOL

CYTOSKELETON

NF-κB

EFFECT OF INHIBITION OF FAK

EFFECT OF INHIBITION OF p38

EFFECT OF INHIBITION OF MSK-1

SIGNALING EVENTS INVOLVED IN SHEAR-INDUCED CHEMOKINE PRODUCTION Phospho-p38

Total p38

Phospho-MSK1

Total MSK1

Phospho-NFκB p65

Total NFκB p65

Phospho-MSK1

Total MSK1

Phospho-NFκB p65

Total NFκB p65

Phospho-FAKTotal FAK

Phospho-NFκB p65

Phospho-p38Total p38

Total NFκB p65

Phospho-FAKTotal FAK

MSK1

Chemokine Production

Fig. 7

A B

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Killingsworth, Rakesh P. Patel, Michael B. Fallon and Akhil MaheshwariHagood, William W. Andrews, Mohammad Athar, Nikolai N. Voitenok, Cheryl

Sadiq S. Shaik, Thomas D. Soltau, Gaurav Chaturvedi, Balagangadhar Totapally, James S.B pathwayκ MAPK-NF-βa FAK-P38

Low-intensity shear stress increases endothelial ELR+ CXC chemokine production via

published online December 31, 2008J. Biol. Chem. 

  10.1074/jbc.M807205200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2009/01/02/M807205200.DC1

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