Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5^cre:ZsGreen^l/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.     

Production of NA by endothelial NO-synthase: an in vitro versus in vivo study

Endothelial-derived relaxing factor (EDRF) is secreted by different endothelia in vivo. It is synthesised by endothelial NO-synthase (eNOS). Despite numerous works, its identity is not fully understood. Here the production of NA, a nitroso-arginine, which was shown to be synthesised by brain NO-synthase (bNOS), was studied in eNOS preparations. NA was quantified by reductive differential pulse voltammetry (RDPV) during its irreversible electrochemical transformation to N-hydroxy-arginine (NHA). Using microelectrodes, NA and nitrite were simultaneously measured in pure recombinant eNOS giving similar enzyme activity. NA was detected at the surface of human endothelial cells (HUVEC) and disappeared when D-arginine was introduced in the culture medium. NA production by endothelium tissue was studied in rat corpus cavernosum using voltammetric microelectrodes. NA concentration at the endothelium surface was linked to vasodilatation measured by laser Doppler induced by acetylcholine injection. LNMA ic injection induced NA disappearance. These preliminary new experiments suggested that NA could be the endogenous nitroso-compound presented early as EDRF.     

Establishment and biochemical characterization of tamarillo (<Emphasis Type="Italic">Solanum betaceum</Emphasis> Cav.) embryogenic cell suspension cultures

Somatic embryogenesis (SE) is an important biotechnological tool with great potential for large-scale cloning. In Solanum betaceum Cav. (tamarillo), embryogenic (EC) and non-embryogenic (non-EC) cells can be obtained from the same explant on auxin-containing medium, making this system ideal for the evaluation of biochemical changes occurring during embryogenic induction. Liquid cultures offer additional possibilities for the analysis of factors controlling SE induction, and the main objectives here were the establishment of cell suspensions and the characterization of the extracellular protein profiles in EC and non-EC cultures. Growth kinetics of liquid cultures, starting with different amounts of EC or non-EC callus or with different weight per volume ratios, were analyzed. Embryogenic suspension cultures were efficiently established starting with 40 mg of cells in 20 mL of liquid medium. Mass spectrometry and fluorometric techniques were employed to identify extracellular proteins, their hydrolytic activity, and the main classes of proteases secreted into the media of EC or non-EC cultures. Extracellular protein profiles revealed quantitative and qualitative differences between EC and non-EC suspension cultures, mainly for several hydrolytic enzymes, such as glucanases and xylanases. Proteolytic activity analysis found serine proteases, aspartic proteases, and metalloproteases in EC cultures, whereas serine proteases were dominant in non-EC lines. For the first time, a protocol for the growth of tamarillo EC and non-EC suspensions was achieved. Moreover, the comparison of protein profiles between EC and non-EC lines showed pronounced differences in the proteolytic and glycolytic enzymes secreted.     

Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth

We have previously hypothesized that the development of severe angioproliferative pulmonary hypertension is associated with not only initial endothelial cell (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC but also with proliferation of vascular smooth muscle cells (VSMC). We have demonstrated that EC death results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype. We postulate here that the initial apoptosis of EC induces the release of mediators that cause VSMC proliferation. We cultured EC in an artificial capillary CellMax system designed to simulate the highly efficient functions of the human capillary system. We induced apoptosis of microvascular EC using shear stress and the combined VEGF receptor (VEGFR-1 and -2) inhibitor SU-5416. Flow cytometry for the proliferation marker bromodeoxyuridine showed that serum-free medium conditioned by apoptosed EC induced proliferation of VSMC, whereas serum-free medium conditioned by nonapoptosed EC did not. We also show that medium conditioned by apoptosed EC is characterized by increased concentrations of transforming growth factor (TGF)-beta1 and VEGF compared with medium conditioned by nonapoptosed EC and that TGF-beta1 blockade prevented the proliferation of cultured VSMC. In conclusion, EC death induced by high shear stress and VEGFR blockade leads to the production of factors, in particular TGF-beta1, that activate VSMC proliferation.     

槲皮素对凝血酶所致的内皮细胞损伤的保护作用研究

本实验采用凝血酶损伤内皮细胞模型,观察了槲皮素对内皮细胞结构和功能的影响．结果表明,槲皮素0．5,5和50umol／L明显降低凝血酶刺激的内皮细胞对内皮素（ET）的释放,增加内皮细胞培养液中组织纤溶酶原激活物（t—PA）量,并可改善内皮细胞的超微结构。     

Nebivolol induces eNOS activation and NO-liberation in murine corpus cavernosum

Erectile function is critically dependent upon the activation of the endothelial nitric oxide synthase (eNOS) in the smooth muscle cells of penile corpus cavernosum tissue. Nebivolol is a β₁-selective β-adrenoceptor blocker (β-ARB) with additional vasodilating properties, which have been attributed to eNOS-activation. Our study investigated whether nebivolol is able to increase eNOS activity in erectile tissue. Murine penile tissue was incubated in an organ bath under control conditions and in the presence of nebivolol or metoprolol. Immunofluorescence staining was performed using specific antibodies against eNOS-activation or eNOS-serine 1177 phosphorylation. Corpus cavernosum smooth muscle tissue was identified using a smooth muscle actin antibody. In addition, slices of murine erectile tissue were incubated with diaminofluorescein (DAF), a specific fluorescence marker for NO-liberation. Under control conditions and after application of metoprolol, we observed a small eNOS-activation and serine 1177-phosphorylation in murine corpus cavernosum tissue. A significant increase in eNOS-activation and serine 1177-phosphorylation of eNOS was observed only in the presence of nebivolol (10 μM). These alterations of the eNOS protein induced after application of nebivolol were associated with a time-dependent increase in DAF fluorescence in murine erectile tissue. We conclude that β-adrenoceptor blockers differentially influence erectile tissue. Since cardiovascular diseases are often associated with the development of erectile dysfunction, the nebivolol-induced eNOS-activation in corpus cavernosum may be beneficial when treating patients suffering from cardiovascular disease.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (50–90% of control) in [³H]thymidine ([³H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.Abbreviations CM conditioned medium - EC endometrial adenocarcinoma - NHEC normal human endometrial epithelial cells     

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.     

Conditioned medium enhances neuritic outgrowth from rat spinal cord explants

Medium conditioned by primary cultures of fetal or neonatal rat skeletal muscle, fibroblasts, or lung cells dramatically increases the neuritic outgrowth from spinal cord explants. After 7 days in vitro, the outgrowth of neurites from 15- to 16-day fetal rat spinal cord slices grown in conditioned medium (CM) covers a 3- to 4-fold greater area than that from slices grown in fresh, nonconditioned (control) medium. Moreover, the pattern of neuritic outgrowth is markedly different in CM-treated slices. In control slices, the neurites form a tangled, dense network of neurites which usually extend only a small distance from the slice edge, while in CM-treated slices, the neurites form a more open network, with the majority of neurites extending radially for long distances (up to several millimeters) from the slice edge. The effect of CM on neuritic outgrowth is not due to a detoxification or modification of the serum in the medium, because increased neuritic outgrowth was observed in slices grown in medium conditioned in the presence or absence of 10% fetal calf serum. The outgrowth-enhancing factor(s) in CM has a high molecular weight, since all outgrowth-enhancing activity is retained by membrane filters with a nominal molecular weight cutoff of 10⁵ daltons. This factor(s) is stable at 58°C for 30 min, and does not appear to be βNGF or fibronectin.     

Immunohistochemical analysis of smooth muscle cells and volumetric density of the elastic system fibers of wild boar (Sus scrofa) penis

The purpose of the present study was to verify the smooth muscle cell distribution and elastic system fibers volumetric density (Vv) in the corpus spongiosum and corpus cavernosum of the wild boar penis. Adult wild boars (n=13) were used. The penile mid shaft fragments were fixed with 4% phosphate buffered formalin solution and/or Bouin's liquid during 24-48 h, and processed using standard histological techniques. The sections were stained with Weigert's Resorcin-Fucsin with previous oxidation. The elastic system fibers Vv was determined in 25 random fields of each fragment using M42 test system. For immunohistochemical analysis, monoclonal anti-alpha actin smooth muscle was used. The histochemical methods detected elastic system fibers in both corpus spongiosum and corpus cavernosum of all animals. The elastic fibers Vv average was 36.6%+/-0.9 for corpus spongiosum and 11.7%+/-0.5 for corpus cavernosum. Through immunocitochemistry, a small quantity of smooth muscle cells was visualized in intimate relationship with blood vessels wall. The great amount of elastic fibers and the smooth muscle cell distribution beneath the endothelium suggest that these fibers may have an important role in penile erection process in the penis of wild boars.     

Efficacy of a cultured conditioned medium of exfoliated deciduous dental pulp stem cells in erectile dysfunction patients

Majority of current treatment strategies against erectile dysfunction (ED) has been consisted of only a supportive care to sustain enough erection during a sexual intercourse. In this study, we investigated whether the cultured conditioned medium of human exfoliated deciduous dental pulp stem cells (SHED‐CM) had an ability to treat ED through fundamentally repairing the pathological damage of vascular endothelial cells of the corpus cavernosum. An open‐label pilot study was performed from April 2016 to October 2020. SHED‐CM was injected directly into the corpus cavernosum of penis of 38 ED patients who visited our clinic and fulfilled the inclusion criteria. Efficacy was assessed using the simplified International Index of Erectile Function (IIEF‐5) questionnaire. The average age and initial IIEF‐5 score of the patients enrolled in this study was 56 (31–79) years old and 13.1 (5–20) points, respectively. Medical history revealed 7 patients with diabetes, 7 patients with hypertension and 1 patient with priapism undergone shunt operation. Of these, 37 patients (97.4%) showed an improvement in IIEF‐5 of an average of 19.3 (7–25) points or 64.4 (10–300) % increase after three injections of SHED‐CM. Eighteen patients (47.4%) achieved more than 21 points (no ED) in IIEF‐5. No adverse events were encountered. This is the first clinical report of ED treatment in the literatures evaluating the efficacy of SHED‐CM. Treatment with SHED‐CM is expected to repair vascular damages of the corpus cavernosum, which are the main cause of ED, and to be widely spread as a fundamental clinical application for ED.     

Anatomy of the baculum-corpus cavernosum interface in the norway rat (Rattus norvegicus), and implications for force transfer during copulation

The baculum is a nonappendicular bone found in the glans tissue of members of five orders of mammals. Its function during copulation is unknown. Anatomical examination of the baculum and corpus cavernosum in the Norway rat (Rattus norvegicus) shows that the two structures are connected by a layer of fibrocartilage, and that the distal tip of the corpus cavernosum swells during erection to surround the proximal end of the baculum. Microradiographs of bacula from sexually experienced males show that regions of the bone may be remodeling; these data suggest that the baculum is load-bearing. On the basis of this anatomy, I propose that the baculum increases the overall flexural stiffness of the penis during copulation by transferring bending and compressive forces from the distal end of the glans to the tensile wall of the corpus cavernosum. Forces on the distal end of the penis during copulation press the baculum against the corpus cavernosum, reducing its internal volume and increasing intracavernosal pressure and corpus cavernosum wall strains. Because the wall of the erect corpus cavernosum is reinforced with inextensible collagen fibers, an increase in wall strain will also increase wall tissue stiffness, and thereby increase the flexural stiffness of the corpus cavernosum.     

Growth cone interactions with a glial cell line from embryonic Xenopus retina

We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.     

Relaxation of canine corporal smooth muscle relaxation by ginsenoside saponin Rg3 is independent from eNOS activation

It has been reported that nitric oxide (NO) is involved in the relaxation mechanism of ginsenoside saponin in various smooth muscle in experimental animals. Although ginsenoside Rg(3) showed both endothelium-dependent and -independent component relaxation in vascular smooth muscle, the action mechanism of the relaxation of corporal muscle is not clear. We, thus, investigated the relaxation mechanism of ginsenoside Rg(3) using isolated canine corpus cavernosum. Ginsenoside Rg(3) concentration-dependently relaxed the canine corpus cavernosum that had been contracted by phenylephrine (PE), in which IC(50) was 1.68 x 10(-5) g/ml. Ginsenoside Rg(3) significantly (P < 0.05) potentiated acetylcholine (ACh)-induced relaxation in endothelium intact corpus cavernosum. Methylene blue (MB) but not N(omega)-nitro-L-arginine methylester (L-NAME) or ODQ (1H-[1,2,4]oxadiazol-[4,3-]quinoxsalin-1-one) modified the dose-response curve of ginsenoside Rg(3). Ginsenoside Rg(3) also significantly potentiated relaxation response to UV light in the presence of streptozotocin (STZ), which was almost completely (P < 0.01) blocked by ODQ. Ginsenoside Rg(3) concentration-dependently inhibited corporal phosphodiesterases (PDE), which resulted in increase of cyclic adenosine monophosphate (cAMP) as well as cyclic guanosine monophosphate (cGMP) contents in corporal smooth muscles. MB inhibited the accumulation of cGMP but not cAMP by ginsenoside Rg(3). These results indicate that mechanism responsible for the relaxation by ginsenoside Rg(3) is not by stimulating endothelial nitric oxide synthase (eNOS) of the canine corporal smooth muscle but by increasing cyclic nucleotide levels through PDE inhibition.     

Microvascular endothelium and pericytes: High yield,low passage cultures

Summary Cultured microvascular endothelial cells (MEC) have become a valuable model for studies of microvascular physiology and pathology. Most current techniques involve manual removal of undesirable cell types or cloning, require one to several months, and yield high population doubling level cultures derived from a relatively small sample of the original population. We have devised a technique to more rapidly produce larger numbers of MEC. This method provided primary cultures consisting predominantly of MEC within 1 wk. The technique involves selective aspiration of gray matter from the bovine cerebral cortex followed by homogenization, sieving, enzymatic dissociation, and then dense plating (10⁴ to 10⁵ vessel fragments/cm²) onto gelatin- or fibronectin-coated plastic. Typical yields were 0.1 to 0.5 × 10⁶ fragments/g of aspirated gray matter. The optimal culture medium for these cells was 15% equine plasma derived serum, 20% conditioned medium, 2% retinal extract, 60% fresh medium, and 500 μg/ml heparin. Cells attached within 24 h, well-spread colonies were present within 1 to 2 d, and cultures approached confluence within 2 to 3 d. Alkaline phosphatase staining confirmed the microvascular origin of the material plated. Morphology, Factor VIII-related antigen staining and 1,1′-dioctacecyl-3,3,3′3,-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein uptake suggested that MEC predominated. Cultures could be passaged and additionally purified by sequential exposure to pancreatin and trypsin-EDTA. Pancreatin selectively removed MEC colonies leaving a relatively homogeneous pericyte population. The relative ease with which such cultures can be produced should facilitate the in vitro study of brain microvascular function and may also provide insights useful for growing MEC from other vascular beds. This work was supported by grants from the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine (American Cancer Soc. IN97G) and from the National Institutes of Health (HL26895 and HL07224), Bethesda, MD.     

Endothelium Expression of Bcl-2 Is Essential for Normal and Pathological Ocular Vascularization

Bcl–2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl–2 (Bcl–2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl–2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl–2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl–2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl–2 allele (Bcl-2^Flox/Flox) and VE-cadherin-cre (Bcl-2^EC mice). Bcl-2^EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl–2 deficient mice. Bcl-2^EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2^EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl–2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl–2 function in the endothelium.     

Communication between the corpus cavernosum penis and the corpus spongiosum penis in a bull diagnosed by modified contrast cavernosography: A case report

Traditional methods of penile cavernosography failed to reveal a vascular defect or shunt from the corpus cavernosum penis of a 6-yr-old Brahman bull with a history of acquired failure of penile erection. When a tourniquet was applied to the penis and the contrast medium was introduced into the corpus cavernosum penis under pressure, however, a communication between the corpus cavernosum penis and corpus spongiosum penis was demonstrated. In a normal bull, cavernosography with the introduction of contrast medium under pressure produced a normal radiographic appearance. It is suggested that modification of accepted techniques of cavernosography to incorporate injection of contrast medium under pressure is useful in diagnosing this particular type of vascular abnormality, and may also aid in the detection of smaller vascular shunts from the corpus cavernosum penis to the superficial penile vasculature.     

Endogenous urotensin II selectively modulates erectile function through eNOS

Background

Urotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor, namely UT receptor. U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear. On these bases the aim of this study is to investigate the mechanism(s) of U-II-induced relaxation in human corpus cavernosum and its relationship with L-arginine/Nitric oxide (NO) pathway.

Methodology/Principal Findings

Human corpus cavernosum tissue was obtained following in male-to-female transsexuals undergoing surgical procedure for sex reassignment. Quantitative RT-PCR clearly demonstrated the U-II expression in human corpus cavernosum. U-II (0.1 nM–10 µM) challenge in human corpus cavernosum induced a significant increase in NO production as revealed by fluorometric analysis. NO generation was coupled to a marked increase in the ratio eNOS phosphorilated/eNOS as determined by western blot analysis. A functional study in human corpus cavernosum strips was performed to asses eNOS involvement in U-II-induced relaxation by using a pharmacological modulation. Pre-treatment with both wortmannin or geldanamycinin (inhibitors of eNOS phosphorylation and heath shock protein 90 recruitment, respectively) significantly reduced U-II-induced relaxation (0.1 nM–10 µM) in human corpus cavernosum strips. Finally, a co-immunoprecipitation study demonstrated that UT receptor and eNOS co-immunoprecipitate following U-II challenge of human corpus cavernosum tissue.

Conclusion/Significance

U-II is endogenously synthesized and locally released in human corpus cavernosum. U-II elicited penile erection through eNOS activation. Thus, U-II/UT pathway may represent a novel therapeutical target in erectile dysfunction.     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

Sumamry A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1×10⁻⁹ M), isobutyl methylxanthine (3.3×10⁻⁵ M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies. This research was supported by grant AG 01312 from the U.S. Public Health Service, Washington, D.C.