Yongxin Gao

Angiotensin type 2 receptor–mediated apoptosis of human prostate cancer cells

Angiotensin II (Ang II) type 1 receptor blocking drugs have been shown to inhibit the growth of prostate cancer cells and delay the development of prostate cancer. Functional Ang II type 2 receptors (AT2R) are present in these cells and inhibit growth induced by epidermal growth factor. The present studies report apoptosis of prostate cancer cells induced by AT2R overexpression. A recombinant adenoviral vector expressing AT2R (Ad-G-AT2R-EGFP) was transduced into prostate cancer cells, including androgen-independent (DU145 and PC3) and androgen-dependent cell lines (LNCaP). Following AT2R transduction, apoptosis was analyzed by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining and caspase-3 activity assays. The results indicate that increased expression of AT2R alone induced apoptosis in the prostate cancer lines, an effect that did not require Ang II. AT2R overexpression in DU145 cells induced inhibition of proliferation, a significant reduction of S-phase cells, and an enrichment of G1-phase cells. The data also indicate that overexpression of AT2R led to apoptosis via an extrinsic cell death signaling pathway that is dependent on activation of p38 mitogen-activated protein kinase, caspase-8, and caspase-3. Finally, the apoptosis induced by AT2R overexpression is partially dependent on the activation of p53, but not on p21. The observations presented here suggest that the ability of increased AT2R expression to induce apoptosis in prostate cancer cells may have potential therapeutic implications for this disease, and suggest that AT2R is a promising novel target gene for prostate cancer gene therapy. [Mol Cancer Ther 2009;8(12):3255–65]

Macrophage migration inhibitory factor in hypothalamic paraventricular nucleus neurons decreases blood pressure in spontaneously hypertensive rats

Macrophage migration inhibitory factor (MIF) expression is increased by angiotensin II (Ang II) within paraventricular nucleus (PVN) neurons of nor‐motensive rats and acts via its intrinsic thiol protein oxidoreductase (TPOR) to counterregulate the central nervous system‐mediated pressor action of Ang II. Considering that the PVN‐mediated actions of Ang II are enhanced in spontaneously hypertensive rats (SHRs) and contribute to the development of hypertension in these animals, we investigated this MIF regulatory mechanism in SHRs. Here, we have demonstrated that Ang II failed to increase MIF protein expression in the PVN of SHRs. Furthermore, although basal levels of MIF protein and mRNA were similar in the PVN of SHRs and normotensive rats, immunostaining revealed that MIF was either absent from or diminished in PVN neurons of SHRs. AAV2‐mediated increases in MIF expression within PVN neurons of young (8 wk old) SHRs produced a chronic attenuation of hypertension and cardiac hypertrophy. However, similar AAV2‐mediated transduction of [C60S]‐MIF, which lacks TPOR activity, did not alter the development of hypertension or cardiac hypertrophy in SHRs. Collectively, these findings suggest that a lack of MIF expression within PVN neurons contributes to the development of hypertension and cardiac hypertrophy in SHRs.—Li, H., Gao, Y., Qi, Y., Katovich, M. J., Jiang, N., Braseth, L. N., Scheuer, D. A., Shi, P., Sumners, C. Macrophage migration inhibitory factor in hypothalamic paraventricular nucleus neurons decreases blood pressure in spontaneously hypertensive rats. FASEB J. 22, 3175–3185 (2008)

Macrophage migration inhibitory factor in the PVN attenuates the central pressor and dipsogenic actions of angiotensin II

Macrophage migration inhibitory factor (MIF) acts intracellularly to counteract the angiotensin (ANG) II type 1 receptor (AT1‐R)‐mediated chronotropic effect of ANG II in hypothalamic neurons, an effect mediated by the thiol‐protein oxidoreductase (TPOR) activity of the MIF molecule. Here we determined the in vivo actions of MIF in regulating the physiological actions of ANG II that are mediated via the paraventricular nucleus (PVN), an area that serves as a relay point in the central nervous system (CNS)‐mediated effects of ANG II on cardiovascular functions and water intake. Intracerebroventricular (icv) injection of ANG II into normotensive rats selectively increased MIF protein levels in the PVN and produced significant pressor and drinking responses that were inhibited by PVN administration of the AT1‐R antagonist losartan. Overexpression of MIF in PVN neurons via Ad‐Syn‐MIF gene transfer attenuated the pressor and drinking responses produced by icv‐injected ANG II. Consistently, intracellular application of MIF or MIF‐(50–65) (which harbors the TPOR activity of MIF) into PVN sympathetic regulatory neurons, blunted the electrophysiological actions of ANG II at these cells. These observations establish for the first time that MIF within the PVN, acting via TPOR, is an intracellular regulator of the central cardiovascular and dipsogenic effects of ANG II.—Li, H., Gao, Y., Freire, C. D., Raizada, M. K., Toney, G. M., Sumners, C. Macrophage migration inhibitory factor in the PVN attenuates the central pressor and dipsogenic actions of angiotensin II FASEB J. 20, E1146–E1156 (2006)

Lack of Macrophage Migration Inhibitory Factor Regulation Is Linked to the Increased Chronotropic Action of Angiotensin II in SHR Neurons

Macrophage migration inhibitory factor acts via its intrinsic thiol–protein oxidoreductase activity to negatively regulate the neuronal chronotropic actions of angiotensin II in normotensive rat neurons. Because the chronotropic action of angiotensin II is potentiated in spontaneously hypertensive rat neurons, we investigated whether this negative regulatory mechanism is absent in these rats. Angiotensin II (100 nM) elicited an ≈89% increase in neuronal firing in Wistar–Kyoto rat hypothalamus and brain stem cultured neurons and an increase in intracellular macrophage migration inhibitory factor levels in the same cells. The chronotropic action of angiotensin II was significantly greater (≈212% increase) in spontaneously hypertensive rat neurons, but angiotensin II failed to alter macrophage migration inhibitory factor expression in these cells. Intracellular application of recombinant macrophage migration inhibitory factor (0.8 nM) or its specific neuronal overexpression via Ad5-SYN-MIF (1×107 infectious units) significantly attenuated the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, similar to results from Wistar–Kyoto rat neurons. In contrast, C60S-macrophage migration inhibitory factor (0.8 nM), which lacks thiol–protein oxidoreductase activity, failed to alter the chronotropic action of angiotensin II in neurons from either rat strain. Thus, whereas macrophage migration inhibitory factor has the potential to depress the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, it is unlikely that this regulatory mechanism occurs, because angiotensin II does not increase the expression of this protein. The lack of this regulatory mechanism may contribute to the increased chronotropic action of angiotensin II in spontaneously hypertensive rat neurons.

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors.

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.

Adenoviral and Adeno-Associated Viral Vectors-Mediated Neuronal Gene Transfer to Cardiovascular Control Regions of the Rat Brain

Viral vectors have been utilized extensively to introduce genetic material into the central nervous system. In order to investigate gene functions in cardiovascular control regions of rat brain, we applied WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) enhanced-adenoviral (Ad) and adeno-assoicated virus (AAV) type 2 vectors to mediate neuronal gene delivery to the paraventricular nucleus of the hypothalamus, the nucleus tractus solitarius and the rostral ventrolateral medulla, three important cardiovascular control regions known to express renin-angiotensin system (RAS) genes. Ad or AAV2 harboring an enhanced green fluorescent protein (EGFP) reporter gene or the angiotensin type 2 receptor gene were microinjected into these brain regions in adult rats. Our results demonstrated that both AAV2 and Ad vectors elicited long-term neuronal transduction in these regions. Interestingly, we found that the WPRE caused expression of GFP driven by the synapsin1 promoter in pure glial cultures or co-cultures of neurons and glia derived from rat hypothalamus and brainstem. However, in rat paraventricular nucleus WPRE did not cause expression of GFP in glia. This demonstrates the potential use of these vectors in studies of physiological functions of certain genes in the cardiovascular control regions of the brain.

627. Adenoviral-Mediated Systemic Transduction of the Angiotensin II (Ang II) Type 2 Receptor (AT2R) Enhances the Hypotensive Action of Losartan

The Ang II type 1 receptor antagonist losartan (LOS) is an effective and specific antihypertensive agent. It has been suggested that a greater beneficial action of LOS may be obtained by co-stimulation of AT2R, which appear to mediate vasodilatory and blood pressure (BP) lowering effects of Ang II. Our overall objective in the present study was to investigate whether systemic over expression of AT2R can enhance the antihypertensive action of LOS in an Angiotensin II-dependent model of high blood pressure. For this study we constructed an adenoviral vector (Ad-CMV-AT2R-EGFP) containing AT2R genomic DNA with two introns and the encoding region. This vector was highly efficient at transducing AT2R in C2C12 myoblasts in vitro, and in liver, heart, kidney and adrenal 3 days following intracardiac injection in vivo. For the experimental protocol, 16 Sprague Dawley rats were infused s.c. via osmotic minipumps with Ang II (200 ng|[bull]|kg|[minus]|1|[bull]|min|[minus]|1) for 3 weeks to produce hypertension. Following this, rats were injected intracardially with either 2.5|[times]|1010 nfectious units of Ad-CMV-AT2R-EGFP or Ad-CMV-EGFP (8 rats/group). At 3 days post viral injections, measurement of mean basal BP via an arterial catheter revealed no differences between the AT2R- (157.9 +/- 6.01 mmHg) and EGFP-treated (150.8 +/-11.11 mmHg) groups. Likewise, basal heart rate was no different between the AT2R and EGFP-treated animals (387.78+/- 49.10 and 349+/-49.36 BPM, respectively). However, the fall in BP produced by LOS (1 mg/kg, i.v.) was significantly greater in the AT2R-treated animals (29.53 +/- 5.94 mmHg) than in the control (EGFP) group (8.9+/-3.97 mmHg), p<0.05. Rats were euthanized immediately following the experiments, and expression analyses revealed the presence of AT2R transcript within the heart and liver of each of AT2R-treated animal. These data indicate that systemic over expression of AT2R is associated with increased anti-hypertensive actions of LOS, and suggest that AT2R over expression may be beneficial in patients treated with AT1R antagonists to lower BP.