The endocrine-gland-derived VEGF homologue Bv8 promotes angiogenesis in the testis: Localization of Bv8 receptors to endothelial cells

Abstract

We recently identified an angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), with selective activity for endothelial cells of endocrine tissues. Here we describe the characterization of a highly related molecule, Bv8, also known as prokineticin-2. Human Bv8 shares 60% identity and 75% similarity with EG-VEGF. The human and mouse Bv8 genes share a common structure. Like EG-VEGF, Bv8 is able to induce proliferation, survival and migration of adrenal cortical capillary endothelial cells. Bv8 gene expression is induced by hypoxic stress. Bv8 expression occurs predominantly in the testis and is largely restricted to primary spermatocytes. Adenoviral delivery of Bv8 or EG-VEGF to the mouse testis resulted in a potent angiogenic response. We have localized the expression of the Bv8EG-VEGF receptors within the testis to vascular endothelial cells. The testis exhibits relatively high turnover of endothelial cells. Therefore, Bv8 and EG-VEGF, along with other factors such as VEGF-A, may maintain the integrity and also regulate proliferation of the blood vessels in the testis.

Figure 1

Human and mouse primary sequences and gene structures. (A) hBv8 sequence. The putative transcription start is indicated by an arrow. The signal sequence is underlined and the residues in red are encoded in an alternative transcript. (B) The predicted amino acid sequences of hBv8, mBv8, and hEG-VEGF. Bv8 isoforms with a 21-aa insert are found in both human and mouse, whereas a corresponding longer form of EG-VEGF has not been identified. Identical amino acids are red. (C) Comparison of h- (upper sequence) and mBv8. Exon 3 encodes the 21-aa insert of an alternate transcript. The coding sequence is in bold. (D) A comparison of ≈2,000 nt of human and mouse promoter regions revealed five distinct blocks of conservation (80–100% identity), representing 615 of 2,000 nt. Arrowheads represent putative hypoxia-response elements (HREs). Boxes are drawn to scale.

Figure 2

Bv8 is predominantly expressed in the testis. (A) Hybridization of RNA dot blots revealed hBv8 signal in testis and peripheral blood leukocytes (PBLs). (B) By Northern blotting, a single 1.8-kb transcript was identified in testis. Equivalent loading was assessed with actin probe (not shown). (C) Bv8 mRNA is detected in late mouse embryogenesis, E17, and in testis, a transcript of 1.8 kb is most abundant. Samples and sizes (in kb) are indicated. TaqMan analysis of human (D) and mouse (E) samples revealed the highest Bv8 expression in the testis in either species, followed by the placenta in human, and by embryo and ovary in mouse. (F) ISH studies revealed restricted expression in adult human (i–iv) and mouse (v–viii) testis within primary spermatocytes. A developmental time course in mouse revealed expression at postnatal day 16, but not at postnatal day 7 or 10 (data not shown). Sense probes did not yield a signal. (G) TaqMan analysis of P19 cultures exposed to normoxic or hypoxic conditions revealed that the Bv8 transcript level increased 320 ± 27%, which is comparable to the 350 ± 15% increase in VEGF over normoxic controls. Data were normalized to GAPDH, and the value for normoxia was arbitrarily set at 100%. The graph represents three experiments. Error bars, SD.

Figure 3

Bv8 induces proliferation, chemotaxis, and survival of ACE cells. (A) Dose–response experiment comparing cell numbers in ACE cultures treated with Bv8 or EG-VEGF; negative control (Ct) was medium without factors. These molecules promote proliferation to a similar extent. (B) The percent apoptotic cells was scored after incubation in serum-free medium (0%), or with addition of 10% FCS, 2 nM (V)EGF, 20 nM (B)v8, or 20 nM (E)G-VEGF. Bv8 or EG-VEGF induced equivalent responses. (C) Bv8 and EG-VEGF induced migration of ACE cells to a similar extent. Error bars, SD. (D) ERK1/2 phosphorylation was assessed by immunoblot (IB) of ACE lysates, unstimulated (C), or treated with VEGF, Bv8, or EG-VEGF. Total ERK1/2 protein level is presented in Lower. (E) After a 15-min stimulation, VEGF, Bv8, and EG-VEGF increased phosphorylated Akt levels. Total Akt was assessed in Lower. In A–E representative experiments are shown.

Figure 4

AvBv8, EG-VEGF, or VEGF induces a potent angiogenic response in the testis. (A) Mouse testes injected with AvLacZ (or PBS; data not shown) demonstrated normal architecture, weight, and overall appearance. (B–D) AvVEGF (B), AvEG-VEGF (C), or AvBv8 (D) increased interstitial capillaries. Evidence of tubular atrophy in many samples suggested an increased interstitial pressure, presumably secondary to the angiogenic response. Note the high content of extravasated product within the interstitium of B–D. Immunohistochemistry for VEGFR-2 shows increased capillary densities in testes injected with AvVEGF (F), AvEG-VEGF (G), and AvBv8 (H). Few capillaries are apparent in the interstitium of control (E). Interestingly, early spermatids are also positive for VEGFR-2. Expression of VEGFR-1 by these cells was reported (40). Arrows indicate endothelium and arrowheads indicate early spermatids. ST, seminiferous tubule; IT, interstitial tissue.

Figure 5

Bv8 receptor expression is restricted to endothelial cells of the testis interstitium. (A and B) ISH with a probe derived from an mR-1 generated specific signal in the interstitium of the mouse testis. (C) Signal is localized to capillary endothelial cells, which are indicated by arrows. A and B are light- and dark-field images, respectively, and C is a higher magnification of the boxed area.