Modulating the functional contributions of c-Myc to the human endothelial cell cyclic strain response.

This study addresses whether pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction in human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC). mRNA and protein expression of c-Myc under physiological (6-10%) and pathological cyclic strain conditions (20%) were studied. Both c-Myc mRNA and protein expression increased 2-3-fold in HUVEC cyclically strained at 20%. c-Myc protein increased 4-fold in HAEC. In HUVEC, expression of mRNA peaked at 1.5-2 h. Subsequently, the effect of modulating c-Myc on potential downstream gene targets was determined. A small molecular weight compound that binds to and stabilizes the silencer element in the c-Myc promoter attenuates cyclic strain-induced c-Myc transcription by about 50%. This compound also modulates c-Myc downstream gene targets that may be instrumental in induction of vascular disease. Cyclic strain-induced gene expression of vascular endothelial growth factor, proliferating cell nuclear antigen and heat shock protein 60 are attenuated by this compound. These results offer a possible mechanism and promising clinical treatment for vascular diseases initiated by increased cyclic strain.

Expression of CYP1A1 and CYP1B1 in human endothelial cells: regulation by fluid shear stress.

AIMS: CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions. METHODS AND RESULTS: We investigated CYP1A1 and CYP1B1 expressions under different durations, levels, and patterns of shear stress. CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity were maximally up-regulated at > or =24 h of arterial levels of shear stress (15-25 dynes/cm2). Expression of both genes was significantly attenuated by reversing shear stress when compared with 15 dynes/cm2 steady shear stress. Small interfering RNA knockdown of CYP1A1 resulted in significantly reduced CYP1B1 and thrombospondin-1 expression, genes regulated by the aryl hydrocarbon receptor (AhR). Immunostaining of human coronary arteries showed constitutive CYP1A1 and CYP1B1 protein expressions in endothelial cells. Immunostaining of mouse aorta showed nuclear localization of AhR and increased expression of CYP1A1 in the descending thoracic aorta, whereas reduced nuclear localization of AhR and attenuated CYP1A1 expression were observed in the lesser curvature of the aortic arch. CONCLUSION: CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR-regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.

Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis.

Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the gamma-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, alpha(V)beta(3), and beta(1) integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and alpha(4)beta(1) while alpha(6)beta(1) contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on alpha(v)beta(3) and alpha(5)beta(1), and alpha(4)beta(1), alpha(6)beta(1), and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration.

Therapeutic neovascularization: contributions from bioengineering.

A number of pathological entities and surgical interventions could benefit from therapeutic stimulation of new blood vessel formation. Although strategies designed for promoting neovascularization have shown promise in preclinical models, translation to human application has met with limited success when angiogenesis is used as the single therapeutic mechanism. While clinical protocols continue to be optimized, a number of exciting new approaches are being developed. Bioengineering has played an important role in the progress of many of these innovative new strategies. In this review, we present a general outline of therapeutic neovascularization, with an emphasis on investigations using engineering principles to address this vexing clinical problem. In addition, we identify some limitations and suggest areas for future research.

Endothelial cell cytochrome P450 1A1 and 1B1: up-regulation by shear stress.

Third-passage human umbilical vein endothelial cells (HUVECs) or fifth-passage human aortic endothelial cells (HAECs) were subjected to 25 dynes/cm(2) for 24 h in a parallel-plate flow system. Matched control cells were maintained in static conditions. Total RNA was isolated and pooled from six to eight slides per experiment. Changes in gene expression were analyzed by Northern blots and reverse transcriptase-polymerase chain reaction. Fold changes were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) values. In HUVECs, arterial levels of shear stress increased mRNA expression of Cytochrome P450 1A1 (CYP1A1) 10.8 +/- 2.1-fold, and CYP1B1 23.1 +/- 3.7-fold; whereas connective tissue growth factor (CTGF) expression was unchanged and endothelin-1 (ET-1) mRNA expression was decreased 0.7 +/- 0.05-fold. The authors determined whether these changes were induced by beta-naphthoflavone, a polyaromatic hydrocarbon, and whether they occurred in HAECs. beta-Naphthoflavone up-regulated CYP1A1 18.3 +/- 4.2-fold, and CYP1B1 4.1 +/- 0.3-fold in HUVECs. Shear stress up-regulated CYP1A1 6.3 +/- 0.4-fold and CYP1B1 51.1 +/- 2.1-fold in HAECs. In addition, the authors examined CYP1A1 and CYP1B1 proteins translated from these genes. Experiments identical to those described above were performed and the cells harvested for protein identification by Western blot of CYP1A1 and CYP1B1. Protein levels of CYP1A1 in HUVECs were up-regulated under shear stress, whereas protein levels of CYP1B1 were not.

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

Platelet aggregation and activation under complex patterns of shear stress.

Arterial stenosis results in a complex pattern of blood flow containing an extremely fast flow in the throat of stenosis and a post-stenosis low flow. The fast flow generates high shear stress that has been demonstrated in vitro to activate and aggregate platelets. One potential problem of these in vitro studies is that platelets are invariably exposed to a high shear stress for a period that is significantly longer than they would have experienced in vivo. More importantly, the role of the post-stenosis low flow in platelet activation and aggregation has not been determined. By exposing platelets to a shear profile that contains both high and low shear segments, we found that platelets aggregate when they are exposed to a high shear stress of 100 dyn/cm(2) for as short as 2.5 s, a period that is significantly shorter than those previously reported (30-120 s). Platelet aggregation under this condition requires a low shear exposure immediately after a high shear pulse, suggesting that post-stenosis low flow enhances platelet aggregation. Furthermore, platelet aggregation under this condition is not activation-dependent because the CD62P expression of sheared platelets is significantly less than that of platelets treated with ADP. Based on these findings, we propose that shear-induced platelet aggregation may be a process of mechanical crosslinking of platelets, requiring minimal platelet activation. This process may function as a protective mechanism to prevent in vivo irreversible platelet activation and aggregation under temporary high shear.

Fully automated three-dimensional tracking of cancer cells in collagen gels: determination of motility phenotypes at the cellular level.

We developed a fully automated three-dimensional cell tracking system that quantified the effect of extracellular matrix components on the infiltration and migration of tumor cells. The three-dimensional trajectories of two highly invasive cell lines, the human HT-1080 fibrosarcoma and the human MDA-MB-231 adenocarcinoma, were determined for long-term infiltration in plain or Matrigel-containing collagen type I gels. We modeled the trajectories with a novel formulation of the continuous Markov chain model that can distinguish between the tendencies for infiltration or lateral motion. Parameters such as the speed of subpopulations, the persistence of motion in certain directions, the turning frequency of the cells, the ultimate direction of motion, and the cell distribution with the infiltration depth were obtained to quantify the migration and infiltration at the cellular level. Distinct migratory and infiltration phenotypes were identified for the two cell types that were significantly dependent on gel composition. The HT-1080 cell line expressed a high motility phenotype on the plain collagen gel surface. The Matrigel-containing gel significantly enhanced the infiltration and the turning frequency of the HT-1080 cells. This study shows that tumor cell infiltration and migration are dynamic processes that depend significantly on the cell type and the microenvironment.

Measurement of the binding forces between von Willebrand factor and variants of platelet glycoprotein Ibalpha using optical tweezers.

BACKGROUND AND OBJECTIVE: Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction. STUDY DESIGN/MATERIALS AND METHODS: Canine GP Ibalpha does not normally bind to human vWf, and thus canine-human GP Ibalpha chimeras were constructed by sequentially replacing human GP Ibalpha structural regions with their canine counterparts. Chinese hamster ovary (CHO) cells, which are frequently used to express platelet GP complexes, were transfected with the chimeric proteins. Optical tweezers (lambda = 830 nm) were used to investigate bond strengths between vWf and different GP Ibalpha canine-human chimeras. Since vWf does not bind GP Ibalpha without high shear stress, the compounds botrocetin and ristocetin were used to induce binding between human vWf and the chimeras. RESULTS: All human-canine GP Ibalpha chimeras bound to vWf in the presence of botrocetin. Replacement of the N-terminal flanking sequence and the first leucine-rich repeat resulted in lower GP Ibalpha/vWf bond strengths than the wild-type human GP Ibalpha/vWf bond strength (P < 0.05). Chimeras lacking the second leucine-rich repeat did not adhere to vWf with ristocetin acting as modulator. CONCLUSION: The N-terminal flanking sequence and the first leucine-rich repeat of GP Ibalpha were found to be important but not necessary for GP Ibalpha to adhere to vWf. The second leucine-rich repeat was found to be critical for GP Ibalpha to bind vWf and could potentially be used in the development of a novel recombinant anti-thrombotic drugs.