Angiotensin-(1-7): a peptide hormone with anti-cancer activity.

The development of peptides as therapeutic agents has progressed such that these small molecules of less than fifty amino acids are currently in use for the treatment of a variety of pathologies. This review focuses on the pre-clinical studies and clinical trials assessing the anti-cancer properties of angiotensin-(1-7) [Ang-(1-7)], an endogenous heptapeptide hormone of the renin-angiotensin system. Ang-(1-7) mediates biological responses by activating mas, a unique G protein- coupled receptor, thereby providing specific targeted actions when used as a therapeutic agent. Studies in in vitro as well as in vivo mouse models demonstrated that the heptapeptide hormone reduced proliferation of human cancer cells and xenograft tumors. This attenuation was concomitant with decreased angiogenesis, cancer associated fibrosis, osteoclastogenesis, tumor-induced inflammation and metastasis as well as altered regulation of growth promoting cellular signaling pathways. In three clinical trials, Ang-(1-7) was well tolerated with limited toxic or quality-of-life side effects and showed clinical benefit in cancer patients with solid tumors. Taken together, these studies suggest that Ang-(1-7) may serve as a first-in-class peptide chemotherapeutic agent, reducing cancer growth and metastases by pleiotrophic mechanisms as well as targeting the tumor microenvironment.

AT1, AT2, and AT(1-7) receptor expression in the uteroplacental unit of normotensive and hypertensive rats during early and late pregnancy.

INTRODUCTION: We investigated the expression of angiotensin receptors in early pregnancy and established whether normal pregnancy or preeclampsia alters the expression and distribution of the uteroplacental AT1R, AT2R and mas/AT1-7R at late gestation. METHODS: The percentage of each receptor subtype present in tissues from virgin rats and from normotensive and RUPP hypertensive pregnant rats was established by in vitro receptor autoradiography. Receptor mRNA levels were determined by quantitative PCR at early and late pregnancy. RESULTS: AT1R mRNA levels were up-regulated in the interimplantation (IIS) site at day 7 of gestation. AT2R mRNA levels were decreased at day 5 and 7 in the IIS but increased in the implantation site (IS) at day 5 and 7 as compared to the IIS at day 5. Mas/AT1-7R mRNA was increased in early pregnancy. In normal pregnancy and RUPP the mRNA for all angiotensin receptors was reduced in the uterus at late gestation. The AT1R accounted for the majority of binding in the uterus of virgin and the placenta of pregnant and RUPP. In RUPP pregnancy there was a significant competition with d-Ala in the placenta labyrinth. DISCUSSION AND CONCLUSION: The expression of angiotensin receptors suggests their involvement in the maintenance of early stages of pregnancy. During late gestation down-regulation of Ang receptors in the uterus may arise from feedback down-regulation by Ang II. In the placenta the levels of AT1Rs are equivalent in the RUPP model. The increased binding of mas/AT1-7R at late gestation in RUPP may represent a compensatory mechanism to reduce uteroplacental vascular resistance.

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague-Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats.

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.

The renin-angiotensin system and the exocrine pancreas.

An accumulating body of evidence strongly indicates a local tissue renin-angiotensin system in the pancreas of a various species. In contrast to the majority of tissues that primarily express the angiotensin type 1 (AT1) receptor, the pancreas is one of the few tissues that contain a significant proportion of the AT2 subtype. Moreover, our findings indicate a greater distribution angiotensin II binding sites in the exocrine pancreas. Although the physiological aspects of a local pancreatic renin-angiotensin system remain largely unexplored, recent studies in our laboratory utilizing an acinar cell model demonstrate both functional AT1 and AT2 receptors. Indeed, we show that the AR42J cell line expresses all components of an angiotensin system including the mRNA for renin, angiotensinogen, angiotensin converting enzyme (ACE), AT1a, AT1b and AT2 receptors. Thus, these cells may be of particular value to study the interplay of the AT1 and AT2 receptors to regulate cell growth and potentially exocrine function.

Analysis of RNA by northern-blot hybridization.

Northern-blot hybridization, also referred to as Northern blotting, is one of several methods developed to detect the presence, to determine the size, and to quantify specific cellular mRNAs. By this method, total RNA or poly(A)(+)mRNA, prepared from the cells or tissue of interest, is fractionated by size on a denaturing agarose gel. The separated RNAs are transferred to a membrane by capillary action or under a vacuum and hybridized to a labeled probe with a base sequence complementary to all or part of the target mRNA. Analysis of hybridization signals determines whether the gene of interest is expressed in the cells or tissue, as well as the size and relative quantity of the target mRNA, if appropriate markers are used.

Angiotensin-converting enzyme expression in human carotid artery atherosclerosis.

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of atherosclerosis in animal models and reinfarction rates after myocardial infarction in humans. Although expression of components of the renin-angiotensin system has been reported in human coronary arteries, no data regarding their presence in carotid arteries, a frequent site for the occurrence of atherosclerosis plaques, are available. The following study sought to determine whether ACE mRNA and protein can be detected in human carotid atheromatous lesions. Twenty-four intact endarterectomy specimens were obtained from patients with severe carotid occlusive disease (17 males and 7 females, aged 68+/-1 years) and fixed within 30 minutes. Carotid artery specimens contained advanced Stary type V and VI lesions, and human ACE mRNA expression and protein were localized in cross sections by the combination of in situ hybridization and immunohistochemistry. Cell type-specific antibodies were used to colocalize ACE to smooth muscle cells, endothelial cells, macrophages, or lymphocytes. ACE protein was localized in the intima, whereas the overlying media was largely free of ACE staining. In less complicated lesions, ACE staining was modest and could be visualized in scattered clusters of macrophages and on the luminal side of carotid artery vascular endothelium. Smooth muscle cells were largely negative. ACE staining increased as lesions became more complex and was most prominent in macrophage-rich regions. The shoulder regions of plaques contained numerous ACE-positive macrophage foam cells and lymphocytes. In these areas, microvessels were positive for endothelial cell and smooth muscle cell ACE expression. However, microvessels in plaques free of inflammatory cells were stained only faintly for ACE expression. Labeling for ACE mRNA mirrored the pattern of protein expression, localizing ACE mRNA to macrophages and microvessels within the intima. In conclusion, atherosclerosis alters carotid artery ACE production, increasing transcription and translation within regions of plaque inflammation. These data provide another important mechanism by which inflammation associated with increased ACE expression may contribute to the progression of atherosclerosis.

Angiotensin II AT1-receptor blockade inhibits monocyte activation and adherence in transgenic (mRen2)27 rats.

This study investigated whether angiotensin II AT1-receptor blockade with losartan inhibits endothelium-monocyte interactions originating from long-term activation of the renin-angiotensin system in hypertensive transgenic rats [TGR(mRen2)27]. The number of circulating activated monocytes, monocytes adhered to thoracic aorta endothelium, and the extent of endothelial cell injury were compared in adult male transgenic (mRen2)27 and age-matched Hannover Sprague-Dawley (SD) rats after 12 days of continuous subcutaneous administration of saline (120 microl/24 h), losartan (10 mg/kg/24 h), or the vasodilator hydralazine (3 mg/kg/24 h). At the doses administered in this experiment, both losartan and hydralazine normalized mRen2 rat blood pressures equal to values in similarly treated SD rats. Compared with saline infusion, administration of either antihypertensive in mRen2 rats reduced (p<0.05) endothelial cell injury, but only losartan significantly (p<0.05) decreased the number of activated circulating and endothelium-adherent monocytes. Infusion of antihypertensives in SD rats had no effect on blood pressures, monocyte activity, or endothelial injury compared with saline administration. These findings suggest that the recruitment and infiltration of leukocytes into the subendothelium associated with renin-angiotensin system-induced hypertension is partly mediated by pressure-independent AT1-receptor pathways.

Estrogen regulation of angiotensin-converting enzyme mRNA.

Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced angiotensin-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma angiotensin II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 21 days of chronic 17beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-1alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.14+/-0.02; renal medulla: 0. 37+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.37 versus 2.49+/-0.59; and aorta: 0.41+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with 1 or 10 micromol/L 17beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor angiotensin II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant angiotensin-(1-7).

Getting started in evidence-based health care: a guide to resources.

In response to the growing interest in evidence-based health care resources, available in print and electronically, a number of new "evidence-based" products have been developed to aid the busy clinician. This paper discusses resources that will assist the librarian in beginning the educational process about evidence-based health care, as well as building a network of informational links that will assist clinicians in evidence-based practice.

In vivo repair of cytosine hydrates in DNA of cultured human lymphoblasts.

Ultraviolet irradiation of DNA in vitro results in the production of a wide variety of pyrimidine base alterations, including cytosine hydrates. Enzymes that initiate the repair of monomeric pyrimidine damage have been identified in both bacterial and mammalian systems; however, the in vivo formation and repair of cytosine photohydrates has not been demonstrated in cellular DNA. Using Escherichia coli endonuclease III as a damage-specific probe, we have shown that ring-saturated pyrimidines are formed in cultured human cells by irradiation with broad-spectrum UV light. In addition, these types of base damage are removed from the DNA of human lymphoblasts within 5 h following the irradiation. Analysis of the action spectrum for the formation of cytosine hydrates in DNA reveals that these photoproducts are formed most efficiently by irradiation in the range of 255-265 nm light, coinciding with the wavelengths that are maximally absorbed by the DNA bases.

Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.

We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.

Formation of purine photoproducts in a defined human DNA sequence.

The formation of DNA base damages by broad spectrum ultraviolet irradiation (250-400 nm) was investigated using a defined sequence of human DNA. The irradiated, 92 base pair, 3'-end of the human alphoid segment was incubated with an enzyme fraction purified from bacteriophage T4-infected E. coli. As previously reported, analysis of reaction products by sequencing gels showed enzymic incision of purine-containing photoproducts as well as pyrimidine cyclobutane photodimers. The purine-incising activity does not require metal ions and was unaffected by beta-mercaptoethanol or dithiothreitol. The formation of the purine photoproducts is independent of buffer; these lesions are produced by irradiation of DNA in Tris, Hepes or phosphate buffers. They are produced at biologically significant wavelengths between 260 to 300 nm. Only low levels were detected above or below this range. The formation of purine photoproducts is dose dependent with similar yields at some specific loci to pyrimidine dimers. These results suggest that purine-containing photoproducts could be of consequence in ultraviolet carcinogenesis.

Wavelength dependence of DNA incision by a human ultraviolet endonuclease.

The wavelength dependence of an ultraviolet irradiation of the DNA substrate for a human endonuclease was determined. Sites of DNA incision for all UVB and UVC wavelengths examined were at cytosines which were neither cyclobutane pyrimidine dimers nor 6-4'-(pyrimidin-2-one)pyrimidines. The optimal wavelengths for formation of these cytosine photoproducts were between 270 and 295 nm. This human endonuclease therefore has a similar ultraviolet substrate specificity to endonuclease III.

Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells.

Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance.

A human endonuclease incises ultraviolet-irradiated DNA at cytosines and oxidized DNA at thymines.

Both ultraviolet irradiation and oxidation of DNA produce a variety of pyrimidine base damages. A human endonuclease recognizes such altered bases on these DNA substrates. This human endonuclease incises ultraviolet-irradiated DNA exclusively at sites of photochemically modified cytosines. The precise sites of incision by the human enzyme were determined by DNA sequencing. Chemically oxidized DNA was incised exclusively at thymine loci. The degree of enzymic cleavage at cytosine photoproducts was identical at each site. However, the extent of incision at selected oxidized thymine residues varied within the DNA sequence. These results indicate that the distribution of thymine oxidative modifications is influenced by the neighboring DNA bases.

Detection of DNA damage in human cells and tissue using sequencing techniques.

Methods have been developed that permit both identification and location of sites of alterations is defined DNA sequences. These methods can be extended to human tissues using the alphoid segment, which comprises 1% of the human genome. This segment can be isolated in ample quantities from human cells and tissues. Once purified and end labeled, this defined segment can be used to detect sites of altered DNA moieties by combining Maxam-Gilbert sequencing protocols with appropriate enzymatic probes and chemical techniques. These studies can be performed in cultured cells or in tissues obtained by surgical excision or autopsy.

Detection of UV purine photoproducts in a defined sequence of human DNA.

The UV-irradiated, 3'-end-labeled, 92-base-pair terminus of the human alphoid sequence was incubated with purified endonuclease v. Previously unreported photoproducts were incised at purine loci. These were not pyrimidine photodimers, 6-4'-(pyrimidin-2'-one)-pyrimidines, base loss sites, or ring-opened purines. Therefore, purine-containing photoproducts, possibly dimers, were incised by the enzyme preparation.

Further purification and characterization of human 3-methyladenine-DNA glycosylase. Evidence for broad specificity.

Further purification of a human placental 3-methyladenine-DNA glycosylase by phosphocellulose column chromatography yielded a 6000-fold increase in specific activity with greater than 5% recovery. Although 3-methyladenine was the predominant base released from double-stranded methylated DNA by this enzyme, minor releasing activities for 7-methylguanine and 3-methylguanine were also observed. During purification, the three DNA glycosylase activities consistently copurified with constant ratios of specific activity. Moreover, all the activities were heat-inactivated at 50 degrees C at the same rate, required double-stranded methylated DNA as substrate, were inhibited by spermine and spermidine, and were not subject to product inhibition. These data strengthen the likelihood that the three activities are associated with a single DNA glycosylase.