Human Genome Screen to Identify the Genetic Basis of the Anti-inflammatory Effects ofBoswelliain Microvascular Endothelial Cells

Abstract

Inflammatory disorders represent a substantial health problem. Medicinal plants belonging to the Burseraceae family, including Boswellia, are especially known for their anti-inflammatory properties. The gum resin of Boswellia serrata contains boswellic acids, which inhibit leukotriene biosynthesis. A series of chronic inflammatory diseases are perpetuated by leukotrienes. Although Boswellia extract has proven to be anti-inflammatory in clinical trials, the underlying mechanisms remain to be characterized. TNF alpha represents one of the most widely recognized mediators of inflammation. One mechanism by which TNFalpha causes inflammation is by potently inducing the expression of adhesion molecules such as VCAM-1. We sought to test the genetic basis of the antiinflammatory effects of BE (standardized Boswellia extract, 5-Loxin) in a system of TNF alpha-induced gene expression in human microvascular endothelial cells. We conducted the first whole genome screen for TNF alpha- inducible genes in human microvascular cells (HMEC). Acutely, TNF alpha induced 522 genes and downregulated 141 genes in nine out of nine pairwise comparisons. Of the 522 genes induced by TNF alpha in HMEC, 113 genes were clearly sensitive to BE treatment. Such genes directly related to inflammation, cell adhesion, and proteolysis. The robust BE-sensitive candidate genes were then subjected to further processing for the identification of BE-sensitive signaling pathways. The use of resources such as GenMAPP, KEGG, and gene ontology led to the recognition of the primary BE-sensitive TNF alpha-inducible pathways. BE prevented the TNF alpha-induced expression of matrix metalloproteinases. BE also prevented the inducible expression of mediators of apoptosis. Most strikingly, however, TNF alpha-inducible expression of VCAM-1 and ICAM-1 were observed to be sensitive to BE. Realtime PCR studies showed that while TNF alpha potently induced VCAM-1 gene expression, BE completely prevented it. This result confirmed our microarray findings and built a compelling case for the anti-inflammatory property of BE. In an in vivo model of carrageenan-induced rat paw inflammation, we observed a significant antiinflammatory property of BE consistent with our in vitro findings. These findings warrant further research aimed at identifying the signaling mechanisms by which BE exerts its anti-inflammatory effects.

Characterization of a preclinical model of chronic ischemic wound

“…To identify clusters of transcripts differentially expressed during the temporal course of healing, the GeneChip high-density microarray approach was adopted (45)(46)(47)(48)(49)(50). Total RNA was extracted (TRIzol, GIBCO-BRL) from wound tissue on 0, 3, 7, 14, and 28 (postheal tissue in the case of closed wounds) days after wounding.…”

“…The quality of RNA thus obtained was examined using the Agilent 2100 Bioanalyzer. Targets were prepared for microarray hybridization according to previously described protocols (45)(46)(47)(48)(49)(50). To assess the quality of hybridization, samples were hybridized for 16 h at 45°C to GeneChip test arrays.…”

“…Tissue mRNA was quantified by real-time or quantitative (Q) PCR assay using the double-stranded DNA binding dye SYBR Green-I as described previously (45)(46)(47)(48)(49)(50). The primer set used for the individual genes are listed below.…”

Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue

“…Amplified RNA was processed as follows: after the second round of cDNA synthesis and purification in the RiboAmp OA protocol, 20 l of eluted cDNA was added to the GeneChip IVT Labeling Kit (Affymetrix, Santa Clara, CA) in vitro transcription reaction to generate biotinylated cRNA. This protocol has been optimized to reproducibly amplify RNA from nanogram quantities of RNA and is based on the principle of performing two cycles of cDNA synthesis and in vitro transcription for target amplification (21). Hybridization.…”

“…The arrays were washed, stained with streptavidinphycoerythrin and scanned with the GeneArray scanner (Affymetrix) in our own facilities as described in refs. [21][22][23][24][25]. Data analyses.…”