Effect of castration on extracellular matrix remodeling and angiogenesis of the prostate gland

This study was conducted to evaluate the long term effect of castration on the prostate gland proliferation, extracellular matrix remodeling and angiogenesis. Prostate gland proliferation was assessed by immunolocalization of proliferating cell nuclear antigen (PCNA). The expression level of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-beta) and metaloprotenase-13 (MMP-13) by the prostate gland were assessed by immunohistochemistry and quantitative real-time PCR. The expression of the above mentioned parameters by the prostate gland of mature intact dogs were compared to that of castrated dogs six months post-castration. The results showed that castration induced a remarkable atrophy of the prostate gland which was associated with a highly significant decrease in the PCNA proliferation index. Although TGF-beta protein was immunolocalized to the epithelial and stroma cells of the prostate gland from both intact and castrated dogs, castration induced a significant up-regulation of TGF-beta mRNA expression. VEGF mRNA expression and its encoded protein immunolocalization were decreased significantly by the prostate gland from castrated dogs as compared to that of intact dogs. Castration, on the other hand, resulted in no significant change in MMP-13 mRNA expression despite an effect on its cellular immunolocalization which appeared to be localized to the epithelial and stromal cells of the prostate gland from castrated dogs as compared to epithelial cells of the prostate gland from intact dogs. These results indicated that castration-induced prostate gland regression continued to exert a potent suppressive effect on prostate gland proliferation which might be mediated by the elevated level of TGF-beta. Moreover, the low expression level of VEGF might reflect a reduced blood flow demand by the regressed and growth-dormant prostate after castration.     

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.     

Altered levels of angiopoietin 1 and tie 2 are associated with androgen-regulated vascular regression and growth in the ventral prostate in adult mice and rats

The involution of the rat ventral prostate gland after castration could be caused by primary changes in the vasculature. To explore the mechanisms, we studied the effects of castration and testosterone treatment on the vasculature in the ventral prostate in adult rats and mice. Androgen receptor expression, vascular morphology, and the expression of angiopoietin (ang) 1 and 2 and their receptor tie 2 were examined 1, 3, and 7 d after castration and after testosterone treatment of castrated animals using stereological methods, immunohistochemistry, laser capture microdissection, and Western blotting. One day after castration, the percentage of blood vessels covered with smooth muscle actin, endothelial cell proliferation, and vascular volume had decreased, whereas endothelial cell apoptosis had increased. Simultaneously, ang 1 and tie 2 protein levels decreased. Nuclear expression of androgen receptor was observed not only in glandular and stroma smooth muscle cells but also in the mural cells of prostate arteries and veins and was markedly down-regulated already 1 d after castration. Testosterone administration of castrated mice and rats reversed all the observed effects. At the mRNA level, tie 2 was exclusively, but ang 1 predominantly, expressed in the stroma, compared with the epithelial compartment. Local delivery of soluble tie 2 during testosterone-stimulated growth, inhibited vascular maturation and increased vascular volume and leukocyte infiltration compared with controls. We conclude that androgens may regulate the prostate vasculature by direct effects on mural vascular cells and by influencing the secretion of the angiopoietins, in above all, the stroma cells.     

Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1

Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1alpha-null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1alpha is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.     

Inhibition of vascular endothelial growth factor in the primate ovary up-regulates hypoxia-inducible factor-1alpha in the follicle and corpus luteum

Vascular endothelial growth factor (VEGF)-dependent angiogenesis is crucial for follicular growth, and corpus luteum formation and function, in the primate ovary. In the ovary VEGF can be hormonally regulated, but in other systems, the main regulator of VEGF expression is hypoxia. We hypothesized that hypoxia was involved in the regulation of angiogenesis in the cycling ovary. We therefore used immunohistochemistry to localize hypoxia-inducible factor (HIF)-1alpha in the marmoset ovary across the ovarian cycle. We also investigated the effect of VEGF inhibition, using VEGF Trap (aflibercept), on HIF-1alpha localization during the follicular and luteal phases of the cycle. Finally, we studied the effect of chorionic gonadotropin stimulation of the corpus luteum during early pregnancy. Nuclear HIF-1alpha staining was largely absent from normally growing preantral and antral follicles. However, there was marked up-regulation of nuclear HIF-1alpha in the granulosa cells at ovulation that persisted into the early corpus luteum. Mature corpora lutea and those collected during early pregnancy had minimal nuclear HIF-1alpha staining. The inhibition of VEGF in the mid-luteal stage resulted in a time-dependent up-regulation of luteal nuclear HIF-1alpha staining (P < 0.05). There was never any nuclear HIF-1alpha in the theca cells of the follicle, but VEGF Trap treatment during the follicular (P < 0.001) or luteal (P < 0.001) phase increased the proportion of antral follicles with nuclear HIF-1alpha staining in the granulosa cells. These results indicate that HIF-1alpha is up-regulated after vascular inhibition, using VEGF Trap, in the follicle and corpus luteum. However, it is also acutely up-regulated during ovulation. This suggests a role for HIF-1alpha in both hypoxic and hormonal regulation of ovarian VEGF expression in vivo.     

Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells

Vascular endothelial growth factor (VEGF) is a main factor promoting neovascularization (angiogenesis) of solid tumours as prostate carcinoma. Hypoxia stimulates VEGF gene expression by activating the hypoxia-inducible factor-1 (HIF-1alpha). In the present study, the hypoxia-mimicking agent Ni(2+) induced vasoactive intestinal peptide (VIP) expression at both mRNA and peptide levels but it did not modify the expression of VIP receptors (VPAC(1), VPAC(2) and PAC(1) receptors) in androgen-dependent human LNCaP prostate cancer cells. VIP increased the mRNA levels of VPAC(1) and PAC(1) receptors whereas it decreased VPAC(2) receptor mRNA level. These features support that hypoxia up-regulation of VIP gene expression in prostatic carcinoma may lead to VIP regulation of the expression of its receptors by means of autocrine/paracrine mechanisms. Either VIP or hypoxia mimetics with Ni(2+) increased VEGF expression whereas both conditions together resulted in an additive response. It suggests two independent mechanisms for the observed pro-angiogenic activities of VIP and hypoxia. VIP did not stimulate HIF-1alpha mRNA expression but increased the translocation of HIF-1alpha from the cytosolic compartment to the cell nucleus. Moreover, VIP was unable to modify the expression of the HIF-1alpha inhibitor FIH-1 discarding the possibility of an indirect effect of VIP on HIF-1 transactivation.     

Biological and Prognostic Significance of the Morphological Types and Vascular Patterns in Colorectal Liver Metastases (CRLM): Looking Beyond the Tumor Margin

Patients with encapsulated colorectal liver metastases (CRLM) have a better prognosis than those without a capsule. The reason for the encapsulation is unknown. Hypoxia inducible factor-1α (HIF-1α) increases tumor angiogenesis and tumor tissue expression is associated with reduced survival. Our aim was to determine whether the good prognosis of encapsulated CRLM is associated with reduced HIF-1α expression by the cancer.The study selected only patients who had not undergone neoadjuvant chemotherapy prior to a potentially curative hepatectomy for CRLM. From 30 selected patients, serial sections were cut from a single randomly selected metastasis. Morphology was assessed following H&E staining. Tumor hypoxia, vascular endothelial growth factor (VEGF), proliferation, and microvascular density (MVD) were assessed by immunostaining for HIF-1α and carbonic anhydrase-9 (CA-9), VEGF, Ki67, and cluster of differentiation-31, respectively. MVD was calculated in the vascular hot spots. Pathology was reported without clinical outcome information. Actual long-term survival was recorded.Thirteen (43%) of the cancers were encapsulated CRLM containing glands which were large, complex, and cribriform. Thirteen (43%) were infiltrative CRLM and their glands were small, closely packed, and rounded with vessels in the interglandular fibrous tissue with no capsule; 3 (10%) had a mixed picture. Encapsulated CRLM had a higher expression of HIF-1α (58% vs 8%, P = 0.03), CA-9 (42% vs 0%, P = 0.04), and VEGF (92% vs 25%, P = 0.02). MVD was lower in the encapsulated CRLM group (37 mm² vs 143 mm², P < 0.001). The median follow-up was 115 months. The encapsulated CRLM group had a better overall and 5-year survival (relative hazard: 0.58, P = 0.057 and hazard ratio: 0.52, P = 0.044).There are 2 main morphological appearances of CRLM which have very different long-term survival following liver resection surgery. The morphology is associated with differences in expression of HIF-1α, CA-9, VEGF, and angiogenesis.     

Estrogen receptor-alpha signaling in growth of the ventral prostate: comparison of neonatal growth and postcastration regrowth

A role for estrogen receptor (ER)-alpha in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ERalpha(-/-) mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ERalpha(-/-) mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ERalpha and FGF10. ERalpha was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ERalpha or ERbeta with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ERalpha agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3-5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ERalpha is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ERalpha can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.     

Early effects of castration on the vascular system of the rat ventral prostate gland

Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.     

缺氧诱导因子1α调控血管内皮生长因子对大鼠缺氧性肺动脉高压的作用

目的　研究大鼠缺氧性肺动脉高压 (HPH)肺血管壁缺氧诱导因子 1α(HIF 1α)和血管内皮生长因子 (VEGF)基因表达的动态变化。方法　 4 0只成年雄性Wistar大鼠随机分成对照组、缺氧 3、7、14和 2 1d组 ,每组 8只 ,测各组大鼠平均肺动脉压 (mPAP)、血管形态学指标、右室肥大指数(RVHI) ;原位杂交和免疫组化检测HIF 1α,VEGF基因表达。结果　缺氧 7d后 ,mPAP开始上升[(18 4 1± 0 37)mmHg],与对照组比较差异有显著性 (P <0 0 5 ) ,缺氧 14d达高水平并维持于此水平。肺血管重塑 ,RVHI改变在缺氧 14d后出现。HIF 1α蛋白在对照组表达不明显 ,各缺氧组肺血管内膜均为阳性 ;在中膜 ,HIF 1α蛋白缺氧 3d始增高 (0 178± 0 0 17) ,与对照组比较差异有显著性 (P <0 0 5 ) ,缺氧 7d达高峰 (0 2 2 1± 0 0 2 1,P <0 0 5 ) ,14d和 2 1d下降 ;HIF 1αmRNA对照组 ,缺氧 3d和 7d组均不明显 ,缺氧 14d增高 (0 2 0 3± 0 0 2 4 ,P <0 0 5 ) ,并维持于高水平。VEGF蛋白在缺氧 7d增高(0 0 74± 0 0 2 2 ,P <0 0 5 ) ,14d达高峰 (0 14 7± 0 0 17,P <0 0 5 )并持续于高水平 ;VEGFmRNA对照组和缺氧 3d组不明显 ,缺氧 7d增高达高峰 (0 138± 0 0 10 ,P <0 0 5 ) ,之后持续于高水平。VEGFmRNA于中膜和内膜、V