Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial-cell-specific mitogen; as such, its role in angiogenesis has been studied extensively. VEGF/VPF may also serve as a local, endogenous regulator of large-vessel endothelial cell integrity. Surprisingly, however, VEGF/VPF expression in normal and/or atherosclerotic vessels has not been previously characterized. Accordingly, we studied normal human arteries and veins as well as atherosclerotic and restenotic human coronary arteries for evidence of VEGF/VPF expression. VEGF/VPF was detected immunohistochemically in sections of normal human aorta, mammary artery, and saphenous vein. Moreover, VEGF/ VPF expression was identified in 32 (97%) of 33 pathological coronary arterial specimens; the extent of VEGF/VPF staining was graded as moderate to strong in 21 of the 32 (66%) positive specimens. VEGF/VPF double immunostaining and in situ hybridization demonstrated that smooth muscle cells constitute the principal cellular source of VEGF/VPF. VEGF/VPF immunostaining among primary atherosclerotic lesions localized predominantly to the extracellular matrix. In restenotic specimens, VEGF/VPF immunostaining was more prominently cellular, particularly among proliferating smooth muscle cells. Although VEGF/VPF expression was observed in areas of macrophage infiltration, double immunostaining failed to localize VEGF/VPF to macrophages in these foci; instead, double immunostaining clearly identified CD45RO-positive cells as responsible for VEGF/VPF expression in such areas. No correlation could be demonstrated between VEGF/VPF immunostaining and extent of vasa vasorum. These findings thus establish that postnatal VEGF/VPF expression is a feature of normal human arteries and veins and is often extensively expressed in arteries narrowed by atherosclerotic plaque. VEGF/VPF expression in the wall and/or plaque of medium to large vessels suggests a role for VEGF/VPF other than promoting angiogenesis. This role may involve maintenance and repair of luminal endothelium.     

血管内皮生长因子及其受体在肝癌细胞中的表达及意义

目的 探讨人肝癌细胞株血管内皮生长因子（VEGF）及其受体的表达,进一步认识VEGF在肝癌血管形成中的作用机制,方法 以人脐静脉血管内皮细胞系ECV304和小鼠成纤维细胞系L929作为对照,采用免疫组化染色及RT-PCR,检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF及其受体的表达。结果 SMMC7721、HHCC和HepG2细胞均有VEGF的表达。同时VEGF受体1（Flt-1)在SMMC7721细胞中也有表达;而HHCC和HepG2细胞则表达VEGF的受体2（KDR）。结论 在肝癌的血管形成中可能存在VEGF的自分泌机制。     

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.     

On the detection of neutrophil-derived vascular endothelial growth factor (VEGF)

In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).     

Differential expression of the angiogenic factor genes vascular endothelial growth factor (VEGF) and endocrine gland-derived VEGF in normal and polycystic human ovaries

Angiogenesis is a key aspect of the dynamic changes occurring during the normal ovarian cycle. Hyperplasia and hypervascularity of the ovarian theca interna and stroma are also prominent features of the polycystic ovary syndrome (PCOS), a leading cause of infertility. Compelling evidence indicated that vascular endothelial growth factor (VEGF) is a key mediator of the cyclical corpus luteum angiogenesis. However, the nature of the factor(s) that mediate angiogenesis in PCOS is less clearly understood. Endocrine gland-derived (EG)-VEGF has been recently identified as an endothelial cell mitogen with selectivity for the endothelium of steroidogenic glands and is expressed in normal human ovaries. In the present study, we compared the expression of EG-VEGF and VEGF mRNA in a series of 13 human PCOS and 13 normal ovary specimens by in situ hybridization. EG-VEGF expression in normal ovaries is dynamic and generally complementary to VEGF expression in both follicles and corpora lutea. A particularly high expression of EG-VEGF was detected in the Leydig-like hilus cells found in the highly vascularized ovarian hilus. In PCOS ovaries, we found strong expression of EG-VEGF mRNA in theca interna and stroma in most of the specimens examined, thus spatially related to the new blood vessels. In contrast, VEGF mRNA expression was most consistently associated with the granulosa cell layer and sometimes the theca, but rarely with the stroma. These findings indicate that both EG-VEGF and VEGF are expressed in PCOS ovaries, but in different cell types at different stages of differentiation, thus suggesting complementary functions for the two factors in angiogenesis and possibly cyst formation.     

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.     

甲状腺癌组织中VEGF和VEGF-C的表达及意义

目的　探讨血管内皮生长因子(VEGF)、VEGF- C在甲状腺癌中的表达及其意义。方法　应用免疫组化S P法检测44例甲状腺癌中VEGF、VEGF C的表达情况,并以17例癌旁正常甲状腺组织作对照。结果　VEGF、VEGF- C在甲状腺癌中呈高水平表达(88. 6%、81. 8% )。VEGF表达随癌组织分化程度的减低而增高, 9例死亡病例均为阳性表达;VEGF- C表达随癌组织分化程度的减低而减低,乳头状癌阳性表达(88 .9% )高于其它类型,VEGF- C阳性率在有淋巴结转移组(92. 0% )明显高于无淋巴结转移组(68. 4% ) (P<0. 05)。9例死亡病例中7例为阳性表达,且7例同时VEGF呈阳性表达。结论　VEGF、VEGF- C表达与甲状腺癌病理分型及预后可能有一定关系,VEGF- C与甲状腺癌淋巴结转移密切相关。     

UV radiation and prostaglandin E2 up-regulate vascular endothelial growth factor (VEGF) in cultured human fibroblasts

OBJECTIVE AND DESIGN: Exposure to UV radiation is responsible for skin erythema and inflammation. PGE2 is an important inflammatory mediator involved in this process and vascular endothelial growth factor (VEGF) is a potent vascular permeability factor mainly produced by epidermal keratinocytes. This study was aimed at determining whether UVB/A1 radiation and prostaglandin E2 (PGE2) could modulate the production of VEGF by cultured dermal human fibroblasts (HF) in comparison to keratinocytes (HK). MATERIALS AND METHODS: The skin cells derived from foreskin, were cultured in defined medium before treatment by either UVB/A1 radiation, or stimulation by addition of PGE2 (10(-8) to 10(-5) M). The expression of VEGF in cultured fibroblasts and keratinocytes was evaluated at the mRNA (RT-PCR) and protein levels (ELISA). RESULTS: The basal level of VEGF was lower in HF than in HK. Both UVB and UVA1 radiation strongly up-regulated VEGF mRNA and protein in HF whereas UVB but not UVA1 radiation induced a VEGF increase in HK only at the protein level. UVA1, when associated with UVB radiation, showed an additive effect on VEGF secretion in HF but not in HK. PGE2 increased in a dose-dependent manner the expression of VEGF in HF but not in HK. Indomethacin as well as the antioxidant alpha-tocopherol did not reduce UV-induced enhanced secretion of VEGF by both fibroblasts and keratinocytes whereas pyrolidine dithiocarbamate exerted an inhibition of this overexpression. CONCLUSIONS: These results indicate different signaling pathways in the PGE2 and UV-induced regulation of VEGF in dermal fibroblasts and epidermal keratinocytes. They also suggest a role for VEGF from both fibroblasts and keratinocytes in the UV-induced erythema, independent of PGE2. A dermal overexpression of VEGF by fibroblasts from UV-irradiated skin may contribute to dilated microvasculature, a feature of skin photoaging and more generally, to a more permissive stroma to tumor formation than unexposed skin.     

Analysis of vascular endothelial growth factor (VEGF) functional variants in rheumatoid arthritis

The vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic mediators related to inflammation-associated synovial angiogenesis. The aim of this study was to asses the role of -1154 G-->A (rs1570360) and -634 G-->C (rs2010963) VEGF gene functional variants with rheumatoid arthritis (RA). The population under study was composed of a total of 753 unrelated RA patients and 801 healthy controls. The VEGF -1154 G-->A and -634 G-->C polymorphism genotyping was performed by real-time polymerase chain reaction technology, using TaqMan 5' allelic discrimination assay. No evidence of association was observed between the -1154 G-->A and the -634 G-->C VEGF polymorphisms, or inferred VEGF haplotypes with RA susceptibility or clinical manifestations. Our results suggest that the analyzed VEGF promoter polymorphisms may not play a relevant role in RA pathogenesis in our population.     

Evaluation of vascular endothelial growth factor (VEGF) in neoplastic and tuberculosis effusions--preliminary results

Angiogenesis is an essential process for tumor progression and VEGF is thought to be a critical factor for this process. VEGF is also known as a strong capillary permeability inducer, what can be important for pleural effusion development. The aim of the study was the comparative analysis of VEGF concentration in pleural effusions collected from 31 patients with various type of cancer, 8 patients with tuberculosis, 5 patients with transudates. Additionally VEGF concentrations in 11 serum specimens from patients with neoplastic disease was evaluated. VEGF concentrations was determined using a sandwich enzyme-linked immunoadsorbent assay. The VEGF level in pleural transudates as well as tuberculous effusions was significantly lower than in malignant fluid. The were no differences in VEGF level in malignant pleural fluid of different origin. The correlation between VEGF concentrations in malignant pleural fluid and sera of individual patients was not found. Our results indicate that VEGF might play an important role in accumulation of pleural fluid especially that of malignant origin. VEGF level in pleural fluid differs significantly from cancer to benign group of patients what can be important for differential diagnosis of plural effusions origin.     

牛蒡子苷调控血管内皮生长因子抑制糖尿病视网膜病变的机制研究

目的探讨牛蒡子苷(Arc)是否通过调控血管内皮生长因子(VEGF)表达进而抑制糖尿病视网膜病变。方法体外培养人视网膜微血管内皮细胞(HRCECs),用不同浓度的Arc处理HRCECs,同时将si-VEGF或pcDNA3.1-VEGF分别转染入HRCECs,MTT法检测HRCECs增殖能力;Western blot检测p21、细胞周期蛋白D1(cyclinD1)、肿瘤坏死因子-α(TNF-α)、人单核细胞趋化蛋白-1(MCP-1)、载脂蛋白M(ApoM)、VEGF蛋白表达。结果与LG组相比,HG组HRCECs的OD值明显升高(P<0.05),cyclinD1、TNF-α、MCP-1、ApoM、VEGF水平均显著升高(P<0.05),而p21表达下调(P<0.05),Arc作用后可显著逆转HG对HRCECs增殖、cyclinD1、TNF-α、MCP-1、ApoM、VEGF及p21表达的作用,且Arc不同剂量组间均存在显著性差异(P<0.05);抑制VEGF表达可明显减弱高糖作用下的HRCECs增殖能力并降低相关炎性因子表达;过表达VEGF能逆转Arc对高糖诱导的HRCECs增殖及炎症反应的抑制作用。结论牛蒡子苷可能通过降低高糖诱导的HRCECs细胞中VEGF的高表达进而抑制HRCECs增殖,并可通过减轻炎症反应保护细胞免受损伤。     

Mast cell-derived vascular endothelial growth factor (VEGF) and microvascular density in diabetic placentae

Inflammation Research -     

Vascular endothelial growth factor (VEGF) in endometriosis

Angiogenesis is likely to be involved in the pathogenesis of endometriosis. According to the transplantation theory, when the exfoliated endometrium is attached to the peritoneal layer, the establishment of a new blood supply is essential for the survival of the endometrial implant and development of endometriosis. From the known angiogenic factors, vascular endothelial growth factor (VEGF) has emerged as a pivotally important regulator of normal angiogenesis and pathological neovascularization. The VEGF protein was evaluated immunohistochemically in the eutopic endometrium of 10 women without endometriosis (group I) at laparoscopy and the eutopic endometrium and peritoneal endometriotic lesions of 43 women with endometriosis (group II). VEGF histological scores were 9.7 +/- 4.3 and 4.0 +/- 2.6 respectively in the epithelium and stroma of the eutopic endometrium of group I women, and 10.3 +/- 2.3 and 3.6 +/- 2.3 respectively in women of group II. In red lesions, the VEGF scores were 11.1 +/- 3.0 in the epithelium and 5.1 +/- 3.0 in the stroma, and in black lesions were 8.6 +/- 2.7 and 1.6 +/- 1.6, respectively. Significantly lower values were observed in black lesions as compared with eutopic endometrium and red lesions, the values of which were similar. Scores were also evaluated according to the phase of the cycle. In eutopic as well as ectopic endometrium, no significant cyclic variations were observed throughout the cycle. However, VEGF content was found to be higher in the eutopic glandular epithelium of women with endometriosis during the late secretory phase, possibly suggesting a more likely tendency to implant. In contrast, significantly higher VEGF content was noted in red lesions as compared with black lesions. During all phases of the cycle, the VEGF content in stromal cells of red lesions was higher than in black lesions. Similarities in VEGF content were observed in the glandular epithelium of the eutopic endometrium of women with endometriosis and red lesions, suggesting that endometriosis probably arises from the peritoneal seeding of viable endometrial cells during retrograde menstruation and that red lesions can be considered as the first stage of implantation. After the attachment phase, the high VEGF levels could provoke an increase in the subperitoneal vascular network and facilitate implantation and viability in the retroperitoneal space. Lower VEGF levels in black lesions explain the decrease in both stromal vascularization, followed by fibrosis and inactivation of the implant.      

Placenta growth factor (PlGF) induces vascular endothelial growth factor (VEGF) secretion from mononuclear cells and is co-expressed with VEGF in synovial fluid

The aims of this study were (i) to determine whether PlGF, VEGF and PlGF/VEGF heterodimers are detected in synovial fluid (SF) and plasma samples from patients with a range of arthropathies; (ii) to describe whether any correlation exists between SF PlGF, VEGF and PlGF/VEGF heterodimer levels and the total and differential SF leucocyte counts; and (iii) to investigate the regulation of peripheral blood mononuclear cell (PBMC) VEGF secretion by stimuli relevant to inflammatory joints. PlGF, VEGF and PlGF/VEGF heterodimer levels were measured in the SF and plasma of patients with a range of arthropathies and normal controls by ELISA. Western blotting for PlGF was performed on SF from three patients with rheumatoid arthritis (RA) and primary inflammatory arthropathies. VEGF was quantified in cell culture supernatants after stimulation with lipopolysaccharide (LPS), PlGF or cobalt ions of PBMC isolated from RA patients and controls. PlGF and VEGF were detected in all SF samples. PlGF/VEGF heterodimers were detected in 10.2% of SF samples, most frequently in RA samples. Western blotting confirmed the presence of PlGF in RA SF. PlGF was detected in 52% of RA and 31% of control plasma samples, and VEGF was detected in 38% of RA and 38% of control plasma samples. PlGF/VEGF heterodimers were detected in 21% of RA samples and none of the control samples. In primary inflammatory arthropathy patients, SF PlGF and VEGF levels correlated significantly with the SF total leucocyte count and the neutrophil count. PlGF was the most potent inducer of PBMC VEGF production in both RA and control subjects. This is the first report of the detection of PlGF and PlGF/VEGF heterodimers in the SF of patients with inflammatory arthropathies, and we have shown for the first time that PlGF up-regulates PBMC VEGF production. PlGF may therefore play a key role in the production of VEGF in the inflammatory joint.     

Levels of vascular endothelial growth factor (VEGF) in serum of patients with endometriosis

BACKGROUND: Elevated concentrations of vascular endothelial growth factor (VEGF) have been detected in the peritoneal fluid of patients with endometriosis. Furthermore, it was postulated that VEGF is involved in the development of endometriotic lesions. The present study is aimed at determining whether high levels of VEGF could also be found in the serum of patients with endometriosis. METHODS: VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum from 131 subjects with surgically confirmed endometriosis and 146 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. Parameters such as demographics, personal habits, menstrual characteristics and clinical profile were collected from each subject included in this study. RESULTS: The mean VEGF levels were not significantly modulated in serum samples of cases compared with controls in a crude general linear model and in a model adjusted for possible confounders. VEGF serum levels did not correlate with the score, stage of endometriosis or the presence of benign gynaecological disorders. However, a correlation was found between circulating concentrations of VEGF and body mass index. CONCLUSION: Although VEGF seems to play a pivotal role locally in the implantation and development of endometriotic lesions, the disease is not associated with a significant modulation in the levels of circulating VEGF.     

Clinical importance of vascular endothelial growth factor (VEGF) for papillary thyroid carcinomas

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and may be produced by some cancer cells. Several recent reports have documented that increased expression of VEGF is associated with risk of recurrence or decreased recurrence-free survival in papillary thyroid cancers (PTC). The aims of this study were to determine whether immunohistochemical expression of VEGF is related to local and distant recurrence of PTC and to evaluate the relationship between hypervascularization and VEGF expression in papillary thyroid carcinomas. VEGF expression was examined immunohistochemically in 48 papillary carcinomas. Ten normal thyroids were used as controls. Patients were followed for 61.7 (range 24-143) months. Twelve of the patients had local and distant recurrences. VEGF immunostaining, blinded for clinicopathological data, was evaluated semiquantitatively by two pathologists. The difference between the recurrent (n:12) and nonrecurrent (n:36) carcinomas was statistically significant (p:0.001). VEGF expression was also stronger in papillary thyroid carcinomas than in normal thyroid tissues. The mean microvascular densities were significantly higher than in normal thyroid tissues. These data indicate that VEGF staining is strongly associated with increased frequency of local and distant recurrence in PTC and that the immunohistochemical profile of the expression may be used as a marker for predicting which tumors have metastatic potential.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

Soluble vascular endothelial growth factor (VEGF) receptor-1 inhibits migration of human monocytic THP-1 cells in response to VEGF

Objective  

We aimed to investigate the regulation and contribution of vascular endothelial growth factor (VEGF) and sFlt-1(1–3) to human monocytic THP-1 migration.     

The role of vascular endothelial growth factor (VEGF) in abnormal vascular changes in the adult rat eye

The aim of this project was to determine if the subretinal delivery of a recombinant adenovirus encoding vascular endothelial growth factor (VEGF) was sufficient to induce changes resembling choroidal neovascularisation (CNV) in a rat model. A recombinant adenovirus was produced encoding vegf164 cDNA (Ad.RSV.VEGF). Transduction of cultured RPE cells confirmed VEGF expression and ensured the absence of Ad.RSV.VEGF-related toxicity. Following subretinal injection into rat eyes, fluorescein angiography indicated that the in vivo delivery of Ad.RSV.VEGF was associated with vascular leakage. Histological analysis demonstrated that changes resembling the early signs of CNV development were also present in the Ad.RSV.VEGF injected eyes. These results suggest that while a transient VEGF expression in the RPE layer is able to induce CNV-related changes, it may be insufficient for the development of a full neovascular membrane. This study demonstrates that virus-mediated gene delivery, in addition to its clinical applications, is a potentially efficient research tool for investigating gene expression-related physiological changes in vitro and in vivo.