Differential Gene Expression Profile in Essential Hypertension

Essential hypertension affects 20-30% of the population worldwide and contributes significantly to mortality and morbidity[1] from cerebrovascular diseases, myocardial infarction, congestive heart failure and renal insufficiency. Essential hypertension is a prevalent disorder that leads to significant morbidity and mortality. Essential hypertension is defined as chronically elevated arterial pressure resulting from an unknown etiology. Intrinsically, it is a complex, heterogeneous, multifactorial syndrome to which environmental factors are partly responsible for. A lineup of aberrant environmental factors, including dietary salt intake[2,3,4], body weight[5,6], physical inactivity[7.8], physical stress[9.10], cigarette smoking [11-14], alcohol consumption[15-18] and inadequate potassium consumption[19,20] contribute to essential hypertension, possibly by infuriating genetically programmed susceptibilities. Results from twins, adoptive and population studies suggest high degree of similarity of blood pressure values, thus indicating the importance of genetic variables in essential hypertension etiology[21-25]. It is assumed that blood pressure is under the control of a large number of genes each of which has only relatively mild effects.

mRNA length [29] .Dot blot analysis is less time consuming, however, no information on the size of the target biomolecules is offered.
DNA microarray technology has the potential to overcome these limitations, as it has allowed unprecedented analysis of thousands of genes in a high-throughput form.On account of its high-throughput expression profiling, DNA microarray technology has become the predominant assay of choice in clinical medicine.This review, to a great extent looks at studies that used gene expression microarray technology in hypertension research to provide information on the disease specific risk profiles and pathology.

Methods
A literature search of the PUBMED database, using the medical headings "hypertension," "blood pressure," "gene expression," and" microarray analysis," will be conducted.The search will include published studies in human beings and as well as experimental models.Additionally, a search will be performed using references cited in original study articles and reviews and if copies of articles cannot be accessed, authors will be contacted.

Methods for the study of gene expression
Methods used to profile gene expression include Northern blot analysis, serial analysis of gene expression, differential display, dot blot analysis, subtractive hybridization and microarray hybridization.

Northern blot analysis
Although more sensitive gene expression techniques have emerged over the last decade, Northern blot analysis remains the standard for detection and quantitation of mRNA.Northern blotting has proven very effective in evaluating the expression levels of troponin c in chicken skeleton and cardiac muscles [30] , arterial natriuretic factor mRNA and peptide in the human heart during development [31] , myosin heavy chain and actin [32] .It is remarkable in that it allows a direct relative comparison of message abundance between samples on a single blot.Regrettably, this technique requires large quantities of RNA and is prone to significant experimental manipulation for each of the genes examined.Taniguchi et al compared Northern blotting analyses with DNA microarrays a n d d i s c o v e r e d N o r t h e r n b l o t t i n g t o b e m o r e s e n s i t i v e a n d c o n s i s t e n t t h a n D N A microarrays [33] .Despite the fact large-scale transcriptome analysis experiments are not performable with Northern blotting, it is however, conveniently used in studies focused on analysis of small numbers of genes.

Serial analysis of gene expression (SAGE)
Serial analysis of gene expression method was recently discovered at John Hopkins University with the intention to create a global picture of cellular function.SAGE enables tagged short sequences of reverse transcribed cDNA to be prepared and identified by DNA sequencing [34] .The SAGE technique can be used to obtain large-scale cardiac gene expression [35,36] .Even supposing the quantitative and cumulative data this technique presents, one limitation is the identification of the genes reported by the SAGE.

Differential Display (DD)
Differential display was first introduced by Liang and Pardee in 1992 [37] .This technique involves the identification and analysis of differentially expressed genes at the mRNA level.The basic principle of differential display is to use short primers in combination with oligo-dT primers to amplify and visualize mRNA in a cell.DD has been a powerful and successful method due to its inherent simplicity to detect changes in mRNA profiles among multiple samples without any prior knowledge of genomic information of the organism studied.

Dot blot analysis
Dot blot is an immunological technique and is a simplification of northern blotting, southern blotting, or western blotting methods [38,39] .This method identifies a known protein in a biological sample.Dot blot differs from western blotting in that protein samples are separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate.The characteristic of dot blot is the use of immunodetection to identify a specific protein.

Subtractive hybridization
Subtractive hybridization is a powerful technique that was first described by Sargent and Dawid for creating cDNA libraries and generating probes of genes expressed differentially [40] .This technique is based on the principle that nucleic acid sequences in common with the two populations can form hybrids.It is the first tool used for identifying differentially expressed genes on a global scale.With Subtractive hybridization, the isolation of genes does not require prior knowledge of their sequence or identity.

Microarray hybridization
Microarray analysis, is a high through-put technique that provides an important tool to study the global patterns of gene expression.Two of the most commonly used microarrays for gene-expression measurements are oligonucleotide GeneChip expression arrays by Affymetrix and cDNA microarrays.Oligonucleotide microarrays contain sets of multiple 25 mer oligonucleotide probes specific for each gene or expressed-sequence tag (EST), whereas cDNA microarrays generally contain longer oligonucleotide probes (usually 25 to 60 bases) or cDNA probes (usually 500 to 1,000 bases) that stand for the specific gene, so cDNA microarrays are more commonly used.It permits quantitative analysis of RNAs transcribed from both known and unknown genes.Microarray analysis is based on the principle of complementary, single-stranded, nucleic acid sequences forming double-stranded hybrids.This technology can simultaneously measure the expression levels of thousands of genes within a particular mRNA sample in a high-through put manner [42,43] .

Research objects
For the DNA microarrays, we could study human or animal models.The spontaneously hypertensive rats (SHR) are the most popular used animal models for essential hypertension, and the Lyon hypertensive rats [44] follows behind, compared to normotensive Wistar Kyoto rats (WKY).

Software tools
The large amount of information generated from microarrays has been a great strength, but is sometimes seen as a frustrating weakness because of the inability to process experimental data easily, assess the data quality, manage multiple data sets and mine the data with userfriendly tools.Most of the microarrays have the suite of software to deal with the results, such as the Affymetrix software for the Affymetrix microarrays.The related software is listed as follows.The software was not designed to do complex statistical analyses and visualization.Rather, it was designed to help the researcher narrow their search from tens of thousands of gene candidates to several hundred or fewer that meet specific, but adjustable criteria.

Altered gene expression in blood
Peripheral blood gene expression has the potential to provide information on underlying pathologic states.Several authors have used whole blood as a surrogate tissue for gene expression in patients with essential hypertension.In their study, Korkor et al identified 49 differentially expressed genes; 31 up regulated and 18 down regulated genes.Amongst genes found to be altered include CD36, SLC4A1, NET1, SESN3, ZNF652, PRDX6, HIP1, FOLR3, ERAP1, CFD [45] .Most of the genes that were differentially expressed were related to immune/inflammatory responses.In a study conducted by Chon et al, gene expression patterns of hypertensives revealed 680 genes that were upregulated as compared to patients who were normotensive on medication [52] .Timofeeva AV et al reported that 22 genes were up-regulated and 18 genes down-regulation in atherosclerotic aorta compared with normal vessel through cDNA microarray, among these, CD53, SPI1, FPRL2, SPP1, CTSD, ACP5, LCP1, CTSA and LIPA genes are up-regulated both in peripheral blood leukocytes from EH patients and in atherosclerotic lesions of human aorta.The majority of these genes significantly positively correlated with hypertension stage as well as with histological grading of atherosclerotic lesions [53] .

Altered gene expression in the tissues and organs
Koo et al reported altered gene expression in the kidneys of adults of spontaneously hypertension rats [54] .Analyzing mRNA from 8-week-old female SHR and age-matched female WKY, 43 up-regulated and 31 down-regulated genes were revealed.
The upregulation of stearoyl-COA desaturase-2 gene and downregulation of taurine/betaalanine transporter gene in SHR compared with WKY rat were reported and in the SHR group, dysregulations of several genes involved in lipid metabolism was also revealed.Seubert et al investigated renal gene expression profiles in SHR and WKY animals at prehypertensiive (3 wk of age) and hypertensive (9 wk of age) stages and identified 22 genes at 3 wk of age and 104 genes at 9 wk of age that were differentially expressed in SHR compared with WKY [55] .There are some other studies identified differential gene expression in animal models of essential hypertension that are listed in table 2.

Results
References 49 genes were found differentially expressed in essential hypertension, 31 up regulated and 18 down regulated.
Korkor et al. (45)  680 genes were found differentially expressed in untreated hypertensives compared to normotensive controls.On the other hand, only 7 genes were differentially expressed in treated hypertensives compared to normotensive controls.
Chen et al. (52)   22 genes were up-regulated and 18 genes demonstrated downregulation in atherosclerotic aorta compared with normal vessel, CD53, SPI1, FPRL2, SPP1, CTSD, ACP5, LCP1, CTSA and LIPA genes are up-regulated in peripheral blood leukocytes from EH patients and in atherosclerotic lesions of human aorta.The majority of these genes significantly (p<0.005)positively (r>0.5)correlated with AH stage as well as with histological grading of atherosclerotic lesions.

Summary and conclusions
Gene expression profiling provides a phenotypic resolution not feasible with standard clinical criteria.Differences in the gene expression profiles found in these studies identify markers useful for diagnostic, prognostic and therapeutic purposes.These findings emphasize the utility of whole blood and target organs as surrogate tissues for gene expression profiling.Gene expression profiling of different animal models of essential hypertension, and comparison of these profiles with human essential hypertension, will assist in determining the complex pathways that comprise the pathobiology of essential hypertension and help with the diagnostic, prognostic and therapeutic purposes in the future.

Table 1 .
Differential gene expression profiling in human blood in essential hypertension.SHR, spontaneously hypertensive rats.WKY, Wistar-Kyoto rats, usually used for the control the experimental group.WTR, wild-type rats.BNR, Brown Norway rats.LHR, Lyon hypertensive rats.SDR, Sprague-Dawley rats.

Table 2 .
Animals' microarray studies utilizing target organ tissue.