Endothelial Cells

Endothelial cells reside at the precise interface of the vessel wall and the circulation to rapidly sense changes in blood flow and respond by secreting or metabolizing vasoactive substances to maintain pressure/flow homeostasis. Furthermore, diverse pathophysiologic factors including biomechanical and humoral stimuli all serve to modulate the functional phenotype of the vascular endothelium [12, 19]. Increasing evidence suggests that biomechanical stimulation plays a key role in

Fig. 10.1 Schematic of the serial analysis of gene expression (SAGE) method. In the first step, cDNA is synthesized from poly (A)+RNA using the biotin-oligo-dT followed by cleavage of the cDNA (Step 2) by the anchoring enzyme (e.g. NlalII), a frequent-cutting (four base-pair recognition sequence) enzyme and expected to cleave most transcripts at least once. The 3'-portion of the cDNA is captured by the streptavidin-coated magnetic beads. Next, two different linkers containing a five base-pair recognition site for a type II restriction enzyme called tagging enzyme such as BsmFI are ligated to two aliquots of the captured cDNA, respectively (Step 3). Short tags plus linkers are then released from the cDNA by tagging enzyme digestion (Step 4), followed by blunting of the ends of the released short tags with the DNA polymerase I, large

(Klenow) fragment. In step 6, the linker-tag molecules are ligated tail to tail to form di-tags which are amplified by PCR. The ditags are released from linkers by the anchoring enzyme digestion and concatenated by ligase (Step 7) and the concatemers are cloned into a sequencing vector and are subjected to DNA sequencing (Step 8). Finally, in step 9, sequencing of concatemer clones reveals the identity and abundances of each tag. Relative abundance can be calculated by dividing the observed abundance of any tag by the total number of tags analyzed to generate quantitative result. The digital data format of transcripts in both normal and diseased samples can be easily analyzed by SAGE software to identify the genes of interest in specific physiologic or disease states. This figure was modified from Ye et al. [9].

the maintenance of vascular integrity as well as in the development of vascular disease [16, 20]. Other analyses of signal cascades, transcriptional factor systems, and the identification of shear-stress-response elements in the promoters of several genes (PDGF-A, VCAM-1, etc.) relevant to the atherosclerotic process [17, 18], have provided insight into the cellular mechanisms linking shear stress stimuli and genetic regulatory events [12, 15]. Garcia-Cardena G. et al. [21] applied transcriptional profiling via cDNA arrays to assess the gene expression in cultured human umbilical vein endothelial cells (HUVEC) exposed to either steady laminar shear stress (10 dynes/cm2) or turbulent or non-laminar shear stress (spatially and temporally averaged shear stress of 10 dynes/cm2) for 24 h. Whereas a greater number of genes were downregulated by either laminar or turbulent shear stress than were up-regulated, direct comparison of the two forms of shear stress revealed 100 genes which were differentially regulated (68 up-regulated; 32 down-regulated, turbulent vs. laminar). The application of laminar shear stress to static monolayers resulted in greater than two-fold changes in 205 genes (from a total of 11,397 unique genes) whereas only 86 genes increased greater than two-fold with turbulent shear stress. These studies clearly indicate that cultured endotheli-al cells can discriminate between these distinct types of fluid mechanical stimulation.

Similar studies in human aortic endothelial cells exposed to laminar shear stress at 12 dynes/cm2 for 24 hours [22] revealed 125 genes (from 1,185 gene arrays), which exhibited significant difference between the sheared and static samples. Genes related to endothelial cell inflammation including MYD-88 (an adaptor protein essential for signaling via the IL-1 receptor), CD30 ligand (a member of the TNF superfamily and an inducer for T-cell proliferation) and platelet basic protein (a chemokine for neutrophils and monocytes) were down-regulated in response to shear stress as detected by microarray analysis and confirmed by Northern blotting. Since exposure of endothelial cells to proinflammatory cytokines such as IL-1 and TNF results in the modulation of "prothrombotic-proinflamma-tory" program, shear stress may antagonize inflammatory responses through gene modulation to prevent the induction of the atherogenic "prothrombotic-proinflam-matory" program. In contrast, genes such as Tie2 (angiopoeitin-1 and -2 receptor), Flk-1 (VEGF receptor) and MMP-1 (matrix metalloproteinase 1) were significantly upregulated, supporting the concept that shear stress promotes endothelial cell survival, angiogenesis [23], migration and wound healing [24].

Additional studies in human umbilical vein endothelial cells exposed to higher levels of shear stress (25 dynes/cm2 for 6 or 24 h) also appear to provide evidence to support the concept that physiological levels of shear stress are protective to en-dothelium [25]. A total of 52 genes were differentially regulated genes (32 up-, 20 down-regulated out of 4,000 genes) in response to shear stress results which were further confirmed by Northern blot analysis. For example, expression of members of the cytochrome P450 (CYP) family of genes (including 1A1 and 1B1) whose gene products are associated with cellular detoxification mechanisms, were dramatically increased. Consistent with this concept, the connective tissue growth factor (CTGF) gene whose product has been postulated to play a role in the develop ment of fibrotic disease [26], and is highly expressed in vascular cells in atherosclerotic lesion, was significantly downregulated [27]. These observations are consistent with the hypothesis that physiological arterial shear stress is protective and retards fibrotic and atherosclerotic disease processes.

Nitric oxide (NO) is a key bioregulatory molecule with expression of endothelial nitric oxide synthase (NOS) regulated by shear stress. Microarray studies have provided mechanistic information as to how shear stress regulates the synthesis of NO in endothelial cells [25] with marked increases in arginiosuccinate synthetase gene expression (2.5 to 3 fold) suggesting that shear stress-induced increase in NO synthesis may dependent on an increase in L-arginine synthesis from L-citrulline. The vasoactive intestinal polypeptide (VIP) receptor precursor gene (1.5 to 2 fold) is also upregulated by shear stress indicating that increased availability of VIP receptors potentially increase VIP-mediated NO production. Similarly, aside from increased NOS expression via shear stress promoter elements, increased elastin gene expression (2 to 3 folds) by shear stress may increase the binding of elastin peptides to the elastin-laminin receptor, leading to an increase in intracellular Ca2+ and activation of eNOS with subsequent NO release.

In contrast to shear stress, there is a paucity of studies in which the effect of cyclic strain on endothelial cell gene expression relevant to normal physiologic and pathophysiologic processes has been examined. We have evaluated the effect of

Fig. 10.2 Scatter Plot on Human Lung Endothelium Gene Expression Exposed to Cyclic Stretch. Expression profiling of HPAEC gene expression after 18% cyclic stretch for 48 h was analyzed using the Affymetrix Microarray Suite software (MAS, ver 5.0). The X axis represents the signal intensity of gene expression in cells exposed to static condition. The

Y axis represents the signal intensity of gene expression in cells exposed 18% cyclic stretch for 48 h. Colored dots represent the level of expression whereas parallel lines represent fold changes (2, 5, 10 and 30 folds). Expression levels high to low correlate with color change from red to yellow.

Fig. 10.2 Scatter Plot on Human Lung Endothelium Gene Expression Exposed to Cyclic Stretch. Expression profiling of HPAEC gene expression after 18% cyclic stretch for 48 h was analyzed using the Affymetrix Microarray Suite software (MAS, ver 5.0). The X axis represents the signal intensity of gene expression in cells exposed to static condition. The

Y axis represents the signal intensity of gene expression in cells exposed 18% cyclic stretch for 48 h. Colored dots represent the level of expression whereas parallel lines represent fold changes (2, 5, 10 and 30 folds). Expression levels high to low correlate with color change from red to yellow.

Fig. 10.3 K-mean Clustering Analysis of Gene Expression in Human Lung Endothelium exposed to 18% cyclic stretch. Expression profiling of HPAEC gene expression after 18% cyclic stretch for varied time intervals was analyzed using GeneSpring (ver. 4.2, Silicon Genetics). The representative clusters representing 1,232 genes are displayed. X axis lists the time points and Y axis represent the normalized intensity. Illustrated clusters identify a unique class of genes that are upregulated at 48 h. Selection of a particular gene (such as CASP9 (487_g_ at, white line), an apoptosis regulator gene (listed in Tab. 10.1) illustrates the expression behavior of the individual gene in a time dependent manner.

Fig. 10.3 K-mean Clustering Analysis of Gene Expression in Human Lung Endothelium exposed to 18% cyclic stretch. Expression profiling of HPAEC gene expression after 18% cyclic stretch for varied time intervals was analyzed using GeneSpring (ver. 4.2, Silicon Genetics). The representative clusters representing 1,232 genes are displayed. X axis lists the time points and Y axis represent the normalized intensity. Illustrated clusters identify a unique class of genes that are upregulated at 48 h. Selection of a particular gene (such as CASP9 (487_g_ at, white line), an apoptosis regulator gene (listed in Tab. 10.1) illustrates the expression behavior of the individual gene in a time dependent manner.

human pulmonary artery endothelial cell exposure to 18% cyclic stretch (CS) on gene expression profiling (Birukov et al. manuscript submitted). Extended cyclic stretch (simulates pathophysiological lung expansion) exposure (48 hr) enhanced thrombin-induced paracellular gap formation and decreased transcellular electrical resistance in conjunction with significant alterations in gene expression detected by Affymetrix human Genechip oligonucleotide arrays. Our preliminary results indicate approximately 60 genes (of ~ 12,000 genes) were upregulated with ~ 140 genes down-regulated (more than 2-fold) following cyclic stretch preconditioning (Fig. 10.2). Fig. 10.3 illustrates the expression profiling of human lung endotheli-um exposed to 18% cyclic stretch for varied time intervals with a representative K-mean cluster (1232 genes) depicted by K-mean analysis which identifies a unique class of genes that are upregulated at 48 h. To assess gene-gene interaction within the cluster, a tree view (dendrogram) analysis was applied and illustrated in Fig. 10.4. Through dendrogram analysis, the related genes within the same cluster, which shows the similar expression pattern, can be identified. Our data reveal that apoptosis-related genes such as DED caspase, DAD-1, CED-3, ICE etc. were among the most up-regulated genes (Tab. 10.1). While experiments utilizing RT-PCR, real-time RT-PCR, Northern and Western blots are currently in progress to validate these data, these data suggest that excessive mechanical cyclic strain promotes programmed cell death and impairs vascular functions.

To identify the repertoire of genes differentially expressed after stimuli associated with the atherosclerotic process, de Waard et al. [28] used serial analysis of gene expression (SAGE) to evaluate quiescent human arterial endothelial cells

Fig. 10.4 A Tree View (Dendrogram) Gene Expression in Human Lung Endothelium Exposed to Cyclic Stretch. Panel A: Expression profiling of HPAEC gene expression after 18% cyclic stretch treatment for 0, 6, 24 and 48 h was analyzed using GeneSpring (ver. 4.2, Silicon Genetics). Approximately 1,232 genes are displayed which allow us to identify the related genes within the same cluster which shows the similar expression pattern. Panel B: Display the adjacent genes of 487_g_ at (Caspase 9) identified in K-mean cluster. The color depicts the raw gene expression levels

Fig. 10.4 A Tree View (Dendrogram) Gene Expression in Human Lung Endothelium Exposed to Cyclic Stretch. Panel A: Expression profiling of HPAEC gene expression after 18% cyclic stretch treatment for 0, 6, 24 and 48 h was analyzed using GeneSpring (ver. 4.2, Silicon Genetics). Approximately 1,232 genes are displayed which allow us to identify the related genes within the same cluster which shows the similar expression pattern. Panel B: Display the adjacent genes of 487_g_ at (Caspase 9) identified in K-mean cluster. The color depicts the raw gene expression levels after CS where the brighter the more expressed. Gray rectangle represents absent of gene. Within the vicinity of CASP9, at least 3 genes (Tissue specific extinguisher, Micro-tube-associated protein 1A/1B light chain 3, and Quinone oxidoreductase homolog) were known to be involved in apoptosis. Inversin, syntaxin 7 and integrin a V may be implicated to have functions related to apoptosis. The hypothetical protein 37884_f_ at) may be indicated to have a functional role in the apopto-sis.

Fig. 10.4 B
Tab. 10.1 HPAEC apoptosis regulatory genes showing upregulation after 18% CS for 48 h

Probe ID

Gene name

48 h

32 746_ at

DED caspase

1.66

33 774_ at

Death trigger

1.53

34892_ at

DR4 homolog

1.36

35 662_ at

New member

1.28

38413_ at

DAD-1

1.50

39436_ at

BNIP 3a

1.56

487_g_ at

CASP 9

1.66

34449_ at

ICE

1.30

33 419_ at

Putative

2.88

treated with oxidized LDL, a strong atherogenic stimulus (6 hrs). Among the 12,000 tags analyzed, ~600 tags (~5%) were differentially expressed representing 56 differentially expressed genes (42 known genes), including the hallmark endothelial cell activation markers such as interleukin 8 (IL-8), monocyte chemoattrac-tant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), plasminogen

Tab. 10.2 List of genes within the same cluster identified by tree view analysis shows similar expression patterns after 18% CS for 48 h

Gene order

Probe ID

Gene name

Fold of expression

1

37132_ at

Inversin

1.59

2

37884_ f_ at

Hypothetical protein

1.58

3

39 370_ at

Microtube-associated protein 1A/1B,

1.57

light chain 3

4

487_ g_ at

Caspase 9 (CASP9)

1.56

5

226_ at

CAMP dependent protein kinase

1.48

6

36079_ at

Quinone oxidoreductase homolog

1.45

7

38 744_ at

Syntaxin 7

1.41

8

39071_ at

Integrin alpha V

1.38

activator inhibitor 1 (PAI-1), Gro-a, Gro-/5 and E-selectin. Differential transcription of a selection of the upregulated genes was confirmed by Northern blot analysis.

An important application of the genomic information obtained by microarray analysis is the potential for novel disease-specific therapeutic approaches. To explore aspects of tumor angiogenesis [29, 30], St. Croix et al. [31] compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of the 170 transcripts predominantly expressed in endothelium, 46 were specifically elevated in tumor-associated endothelium including extracellular matrix proteins (such as several collagens: type IV, a2; type VI, a1 and a2; CD146), while many genes remain with an as yet unknown function. These studies provide molecular information on tumor biological processes, which may have significant implications for the development of anti-angiogenic therapies. In fact, data obtained from St. Croix's group have been further examined by Novatachkova et al. [32] concluded that up-regulated transcripts in angio-genesis are involved in extracellular matrix remodeling, cellular migration, adhesion, cell-cell communication rather than in angiogenesis initiation or integrative control. These studies strongly suggest potential application of microarray in therapeutic approaches.

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Detox Diet Basics

Detox Diet Basics

Our internal organs, the colon, liver and intestines, help our bodies eliminate toxic and harmful  matter from our bloodstreams and tissues. Often, our systems become overloaded with waste. The very air we breathe, and all of its pollutants, build up in our bodies. Today’s over processed foods and environmental pollutants can easily overwhelm our delicate systems and cause toxic matter to build up in our bodies.

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