Domestic Animal Endocrinology 48 (2014) 100–109

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Isolation of endothelial cells and pericytes from swine corpus luteum G. Basini*, I. Falasconi, S. Bussolati, S. Grolli, R. Ramoni, F. Grasselli Dipartimento di Scienze Medico-Veterinarie, Università degli Studi di Parma, Parma, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 October 2013 Received in revised form 25 February 2014 Accepted 26 February 2014

From an angiogenesis perspective, the ovary offers a unique opportunity to study the physiological development of blood vessels. The first purpose of this work was to set up a protocol for the isolation of pig corpus luteum endothelial cells, which were characterized by both morphologic parameters and the expression of typical molecular markers; we also verified their ability to form capillary-like structures in a 3-dimensional matrix, their response to hypoxia and their migration in the presence of vascular endothelial growth factor (VEGF). The effectiveness of our isolation protocol was confirmed by the characteristic “cobblestone shape” of isolated cells at confluence as well as their expression of all the examined endothelial markers. Our data also showed a significant cell production of VEGF and nitric oxide. Isolated endothelial cells were also responsive to hypoxia by increasing the expression and production of VEGF and decreasing that of nitric oxide. In the angiogenesis bioassay, cells displayed the ability of forming capillary-like structures and also exhibited a significant migration in the scratch test. Our data suggest that the isolation of luteal endothelial cells represents a promising tool in experiments designed to clarify the biology of the angiogenic process. Furthermore, we have demonstrated that the isolated population comprises a subset of cells with a multidifferentiative capacity toward the chondrocytic and adipocytic phenotypes. These data suggest the presence of a perivascular or adventitial cell niche in the vascular wall of the corpus luteum populated with cells showing mesenchymal stem cell-like features, as already demonstrated for the adipose tissue and endometrium. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Angiogenesis VEGF NO Ovary Pericytes

1. Introduction Two processes are responsible for the development of new blood vessels and both result in the formation of simple, endothelium-lined capillary-like tubes: vasculogenesis, which is de novo differentiation of blood vessels from mesodermal precursor cells, and angiogenesis, the formation of new blood vessels by migration and proliferation of endothelial cells from preexisting vessels [1]. This is a tightly regulated process, involving a balance between a plethora of pro- and anti-angiogenic factors [2]. Vasculature in the adult

* Corresponding author. Tel.: þ39 0 5210 32775; fax: þ39 0 5210 32770. E-mail address: [email protected] (G. Basini). 0739-7240/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2014.02.007

organism is normally in a quiescent phase except for the reproductive tract: in fact, many aspects of reproduction, from cyclic changes in the ovary to implantation and placental function are dependent on physiological angiogenesis. In particular, changes occurring after ovulation include dramatic growth and vascularization of the ovulated follicle, which gradually develops into a mature corpus luteum (CL) [3]. The growth rate of the early developing CL is so rapid that it can be compared with that of extremely aggressive tumors, and it is associated with a rapid increase in luteal vascularity [4]. Corpus luteum development appears strictly dependent on the formation of an extensive capillary network, and impairment of angiogenesis is usually associated with an abnormal luteal function [5–8]. Given that endothelial cell population is relevant within the corpus luteum [5,9] and plays a critical role in its formation

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and function, we have undertaken this research to set up an isolation protocol for swine luteal endothelial cells, whose functional characterization is still lacking in scientific literature. While the presence and function of endothelial cells from the bovine corpus luteum are well documented [10,11], the development of a pig model would represent an important tool to study the biology of angiogenesis, given that swine endothelial cells are widely used for gaining insight on a variety of human pathologic conditions [12–14]. After cell isolation, the reliability of the protocol was verified by means of a morphologic cell characterization; we also studied the expression of a series of markers typical of endothelial cells (CD31, CD105, VE-cad, Cav-1, vWF, VEGF, Flt-1, and KDR) [15–19] as well as the cell potential to organize capillary-like structures in a tridimensional matrix [20]; the effect of hypoxia, a well-known angiogenesis stimulus and the cell migration in the presence of vascular endothelial growth factor (VEGF) [21] were also assessed. Recent evidence [22] suggest that pericytes, mesodermally derived stem cells that wrap around the outside of capillaries, could play a role in the regulation of angiogenic vessel formation; on this basis, a final purpose of our work has been to verify the presence of these cells within the pig corpus luteum and to assess their osteogenic, chondrogenic, and adipogenic potentials [23]. 2. Materials and methods All reagents, unless otherwise indicated are produced by Sigma Chemical Co Ltd (St. Louis, MO), except for endothelial cell culture medium, EBM-2 (Clonetics, Lonza, Walkersville, MD). 2.1. Isolation and culture of endothelial cells from swine corpus luteum Swine ovaries were collected at the slaughterhouse from crossbred gilts whose stages of estrus cycle were unknown. Isolation of endothelial cells was carried out as described by Spanel-Borowski and van der Bosch [24] The last cell suspension obtained was filtered with a sterile gauze (150 mesh) and then using a 70 mm filter (BD Falcon, Bioscience, Bedford). To eliminate the red blood cells, the suspension was treated with 0.17 M NH4Cl for 1 min and centrifuged (500  g for 10 min). At the end of the isolation protocol, 500 mL of cell suspension were plated in 25 cm2

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flasks with 5 mL EBM-2; every 48 h, a change of the culture medium was performed thus eliminating not adherent cells. 2.2. Characterization of endothelial cells The isolated cells were subjected to 3 types of characterization:  morphologic, mainly based on the microscopic observation of cells;  molecular, focused on the assessment of the expression of different endothelial markers;  functional, to verify the ability of the isolated cells to perform typical activities of endothelial cells.

2.2.1. Morphologic characterization Cells were seeded in 5 mL of EMB-2 in 25 cm2 flasks and incubated at 37 C in 5% CO2. After 24 h, flasks were rinsed twice with PBS to remove nonadherent cells and the medium changed. Culture medium was the renewed every 48 h, until the complete confluence. 2.2.2. Molecular characterization The expression of typical endothelial cell markers was verified by reverse-transcription polymerase chain reaction (RT-PCR) (Table 1). As a control, we used a line of endothelial cells from porcine aorta aortic endothelial cells (AOC), kindly provided by Professor Jose Yelamos (Hospital Universitario Virgen de la Arrixa, El Palmar, 30,120 Murcia, Spain) grown in medium M199 with 20% FBS. RNA extraction was performed using NucleoSpin RNA II kit (Macherey-Nagel GmbH, Duren, Germany) according to the manufacturer’s instructions, starting from confluent flasks containing approximately 1.5  106 cells. Complementary DNA (cDNA) was then derived by reverse transcription of 1 mg of total RNA with the kit High-Capacity cDNA Reverse Transcription (Applied Biosystem Inc, Foster City, CA). Finally, the cDNA was amplified with primers specific for each marker using the thermal cycler PTC-100 (MJ Research, Waltham, MA). Each sample was prepared by mixing 1 mL dNTPs (10 mM each), 0.25 mL forward primers (25 mM), 0.25 mL reverse primers (25 mM), 5 mL cDNA, 0.5 mL Dream Taq polymerase (5 UI/mL, Fermentas, Hanover, MD), all brought to a final volume of 50 mL with

Table 1 Gene data and polymerase chain reaction conditions. Marker Forward primer gene CD31 CD105 vWF VE-cad Cav-1 VEGF KDR

50 -AAA AAC CTC CAG TAC ATT TC-30 50 -GCC GAG GAC ACA GAT GAC AA-30 50 - AAT ATC GGC CCT CAG CTC ACCC-30 50 -GAC TCA TCC GAC TCT GAC A-30 50 -ATC CGG GAA CAG GGC AAC ATC-30 50 -AGC TAC TGC CGT CCA ATC GAG-30 50 -GAG TGG CTC TGA GGA ACG AGT G-30

Flt-1

50 -GTA AAA ATG CTG AAA GAG GG-30

Reverse primer

Amplicon Gene bank Cycle Annealing accession number number temperature length or reference (bp) ( C)

50 -ACA GGT GTA TGT CCC GCT GT-30 50 -TCC TGG GAC GC AGG GCT ACG-30 50 -AGA TCC CCA GGC TTC CTC TC-30 50 -CCC AGA CAG AAC ACC ATC CT-30 50 -GCC TCA AAG AGT GGG TCA C-30 50 -TGT CAC ATC TGC AAG TAC GTT CG-30 50 -AAC AAA CCT CTT TTC TGG ATA CCT CG-30 50 -GCC ATC CAC TTC AGA GGA AG-30

35 35 35 35 35 30 35

55 55 55 58 55 60 55

686 484 512 355 434 415 313

X98505 NM_214031.1 BK007995 [17] NM_214438.2 [18] [18]

42

50

623

XM_001925740.4

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Fig. 1. Endothelial cells isolated from swine corpus luteum cultured in EBM-2. The photos are representative of 6 (A), 12 (B), 18 (C) and 24 (D) d of culture.

diethylpyrocarbonate water. The program of the thermal cycler has been so set: preinitial denaturation at 94 C for 2 min, followed by consecutive cycles consisting of denaturation at 94 C, annealing at specific temperatures depending on the primer used (Table 1) and elongation at 72 C and finally a further elongation at 72 C for 10 min. Then, the products of the previous reaction were run on agarose gel at 1.5% (USB Corporation, Cleveland, OH) in Trisborate-EDTA buffer, with the addition of 2 mL of Gel Red (Biotium, Hayward, CA). 2.2.3. Functional characterization This type of characterization was carried out taking into account the following aspects:  formation of capillary-like structures in an in vitro angiogenesis assay;  production of angiogenic factors;  response to hypoxia;  migration ability. 2.2.3.1. In vitro angiogenesis assay. This assay was performed as described by Basini et al [25]. Initially, the cells were cultured in flasks with EBM-2 medium until reaching the confluence. The microcarriers (MCs) (12.5 mg) were then incubated in PBS for 3 h to hydrate. After 3 washings in the medium, the MCs were placed in flasks containing 5 mL of EBM-2 medium with 2% serum together with the detached cells. After 1 night incubation, the fibrin gel was prepared. MCs (20 mL) covered by the cells were pipetted into 6 well plates containing a solution of fibrinogen (1 mg/mL PBS, pH 7.6), added with 1250 IU thrombin (250 mL). Plates were incubated for 30 min at 37 C, then the gels were equilibrated for 60 min with 2 mL of EBM-2 with 2% serum. After a change of medium, plates were incubated at 37 C under

humidified atmosphere (5% CO2); every 48 h the medium was renewed. Because a capillary-like structure is composed of at least 3 endothelial cells and should reach a length of at least 200 mm [26], after 5 d from the polymerization of the gel, the newly formed extensions were stained with Hoechst 33,258, a fluorescent nuclear dye and measured using the micrometer present on the eyepiece of the microscope. 2.2.3.2. Evaluation of the response to hypoxia. Cells (2  105) were seeded in 500 mL EBM-2 (7% FCS), without VEGF, in a 12 well plate to study the effect of hypoxia on the production of VEGF and nitric oxide (NO); to evaluate the expression of VEGF approximately 5  105 cells were plated in 25 cm2 flasks, and the RNA was extracted only when reaching confluence. The cells were incubated at 37 C in an atmosphere of 5% CO2. After 24 h and 48 h for the plates and the flasks respectively, cultures were carried on for 18 h in normoxia (19% O2) and hypoxia (5% O2) by using Anaerocult C mini (Merck KGaA, Darmstadt, Germany). At the end of 18 h, the plates were centrifuged to collect supernatants, used for the determination of NO and VEGF. 2.2.3.2.1. Expression of VEGF in response to hypoxia. The cells were detached from the flasks and RNA was extracted. Subsequently, the reverse transcription was performed and the obtained cDNA was amplified by means of a multiplex polymerase chain reaction, variant of polymerase chain reaction in which 2 or more loci are simultaneously amplified in the same reaction. In this case, the housekeeping gene actin, which is amplified together with the gene of interest was chosen as a control. For the amplification of swine actin, pACTIN sense primers (50 -GAG ACC TTC AAC ACG GCC-3 0 ) and pACTIN antisense (50 -GGA AGG TGG ACA GCG AGG-30 ) (MWG Biotec, Ebersberg, Germany)

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Fig. 2. Expression of typical endothelial cell markers in endothelial cells isolated from swine corpus luteum and in swine aortic endothelial cells (AOC), used as a control.

were used that give an amplified equal to 685 bp and for the VEGF amplification the primers shown in Table 1 were used. The polymerase chain reaction mixture (50 mL) was prepared mixing 5 mL Taq Buffer, 1 mL dNTPs (10 mM), 0.5 mL Dream Taq polymerase (5UI/mL), 1 mL VEGF sense primer (25 mM), 1 mL VEGF antisense primer (25 mM), 0.5 mL pACTIN sense primer (25 mM), 0.5 mL antisense primer pACTIN (25 mM), 5 mL cDNA, all brought to a final volume of 50 mL with diethylpyrocarbonate water. The reaction products were subsequently run on agarose gel at 1.5% and the resulting bands were analyzed with the program Image J (http://rsb.info.nih.gov/ij/). The intensity of the bands of VEGF expression has been

normalized with respect to the levels of expression of the bands of actin used as an internal control for each individual sample. 2.2.3.2.2. VEGF assay. VEGF levels were quantified by means of enzyme-linked immunosorbent assay kit (Quantikine, R & D Systems, Minneapolis, MI) validated for pig [27]. The color reaction developed was read at 450 nm using the Victor Multilabel Counter3 (Perkin Elmer, Boston, MA). 2.2.3.2.3. NO assay. NO production was evaluated by the Griess test that allows to measure the levels of nitrite in conditioned media. The colorimetric reaction developed was measured at 540 nm with the aid of the spectrophotometer Victor Reader.

Fig. 3. (A) Microbeads coated with EC stained with Hoechst and visualized with a fluorescence microscope (10). (B) Microbeads coated with EC displayed with phase contrast microscope (10).

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2.3.1. Osteogenic differentiation To achieve this differentiation, the medium was supplemented with [29] dexamethasone (100 nM), ascorbic acid (0.25 mM), and glycerol-2-phosphate (10 mM). The differentiation medium was changed every 3 to 4 d and staining was performed after about 10 d. The slides were washed in sterile PBS, and fixed in paraformaldehyde (4%) at 4 C for 30 min. The staining method of Von Kossa was used [30], which allows to visualize the deposition of salts of calcium phosphate produced by osteocytes, as described by Donofrio et al [31]. 2.3.2. Adipogenic differentiation Once the cells have reached confluence, they were incubated for 3 d in EBM-2 (7% FCS) added with a mix of substances [32] represented by insulin (1.7 mM), indomethacin (0.2 mM), dexamethasone (1 mM), and isobutylmethylxanthine (0.5 mM). Then, cells were cultured for 24 h in a maintenance medium consisting of EBM-2 (7% FCS) with insulin (1.7 mM). The 2 different media were alternated for 3 cycles. At the end, cells were stained with the method Oil Red O [33]. Fig. 4. Effect of hypoxia on VEGF. (A) Gel representative of the effect of hypoxia on the expression of VEGF. 1: Normoxia; 2: Hypoxia (5% O2). (B) The graph presents the mean  SEM of the optical densities of the bands of VEGF normalized with respect to those of the respective actins. Different letters indicate significant difference (P < 0.05). SEM, standard error of the mean; VEGF, vascular endothelial growth factor.

2.3.3. Chondrogenic differentiation Once reached confluence, cells were treated with EBM-2 (7% FCS) containing a specific supplement for

2.2.3.3. Assay of migration: scratch test. To test the ability of endothelial cell to migrate, the so-called “scratch test” [28] was used. The cells were seeded at a density of 2  104 cells/cm2 in 12-well plates with medium EBM-2 (7% FCS) with or without VEGF. Once reached an 80%– 85% confluence, the medium was replaced using EBM-2 (7% FCS) containing VEGF (Peprotech, Rocky Hill, NJ) at a concentration of 25 ng/mL or 100 ng/mL. Next, using a sterile pipette tip, a scratch of about 1 mm was produced, on the surface of cell monolayer, removing a strip of cells. It was then assessed the ability of these cells to migrate, making photographs of the same visual fields at intervals of 0 to 4, 4 to 8, 8 to 12 h from scratch. The photographs were processed by placing a line intercepting a minimum of 10 cells on each edge of the scratches over time. To quantify cell migration, the size variations of the scratch during the spent time were calculated and reported as a percentage using the size of the scratch at time 0 as a maximum percentage (100%).

2.3. Checking for the presence of pericytes Because a recent work [23] suggests that pericytes, with mesenchymal stem cell capacity, reside in the perivascular microenvironment, we proceeded to test the presence of such cells by trying to induce their differentiation into osteogenic, adipogenic, and chondrogenic phenotypes; for this purpose the cells (15  103/well) were seeded in 6-well plates in EBM-2 (7% FCS). Once reached confluence, different treatments were administered.

Fig. 5. Production of VEGF (A) and NO (B) by endothelial cells under hypoxic conditions (5% O2). Data are expressed as mean  SEM. Different letters indicate significant difference (P < 0.05). NO, nitric oxide; VEGF, vascular endothelial growth factor.

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Fig. 6. Representative phase contrast microscope images (10) of cell migration and invasion of the scratch in the well containing EMB-2 7% FBS with VEGF 25 ng/mL (A) or 100 ng/mL (B). VEGF, vascular endothelial growth factor.

chondrogenic differentiation (STEMPRO chondrogenesis differentiation kit, Gibco, Grand Island). This medium was renewed every 3 d. After 10 d, some groups of cells tended to detach from the bottom, so it was decided to proceed with the staining. The cell clumps were harvested and centrifuged to obtain a pellet. At the end the samples were fixed in formaldehyde (4%) for 30 min, included in agarose (2%), and finally embedded in paraffin. Sections (5 mm) were processed with the Masson trichrome stain according to Passos et al [34].

2.4. Statistical analysis Each experiment was repeated at least 5 times with 6 replicates for each treatment. The data are presented as mean  standard error of mean. In all experiments, statistical differences were calculated by analysis of variance using the software Statgraphics Plus 5 (STC Inc, Rockville, MD). When a significant difference (P < 0.05) was found, means were submitted to the Scheffé F test for multiple comparisons.

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Fig. 7. Endothelial cells after 10 d of treatment for osteogenic differentiation. (A) Control, (B) cells undergoing differentiation. Optical microscope images (4). The arrow indicates a cluster of cells.

3. Results

3.1.3. Functional characterization

3.1. Characterization of endothelial cells

3.1.3.1. In vitro angiogenesis assay and response to hypoxia. After 5 d from gel set up, 1 to 3 elongations longer than 200 mm in each microbead were measured and the multicellularity of these structures was verified (Fig. 3).

3.1.1. Morphologic characterization The first growing cells adhering to the bottom of the flask were observed after 2 to 3 d from plating. After 6 d, the cells began to cluster, forming colonies around d 12, a typical behavior of endothelial cells. After 15 d, at full confluence, the cells displayed the typical endothelial cell shape with a “cobblestone” effect (Fig. 1). 3.1.2. Molecular characterization The expression of the typical endothelial cell markers described in Table 1 was demonstrated by RT-PCR and electrophoretic analysis. A line of endothelial cells from porcine aorta (AOC) was used as control. Figure 2 shows the presence of the considered markers.

3.1.3.2. Evaluation of the response to hypoxia. The expression of VEGF (Fig. 4) was significantly (P < 0.05) stimulated by hypoxia (5% O2) as well as its production (Fig. 5A). On the contrary, hypoxia inhibited (P < 0.05) NO production (Fig. 5). 3.1.3.3. Assay of migration: scratch test. Cells did not migrate in the absence of VEGF while a significant migrating ability was observed only in the presence of VEGF; the extent of the migration, however, did not differ in the presence of different VEGF concentrations tested (Fig. 6).

Fig. 8. Optical microscope images (20 and 40) of (A) control cells and (B) cells undergoing adipogenic differentiation after Oil Red O staining (the arrows indicate the red spots that highlight the vacuoles of fat).

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Fig. 9. Sections of clumps formed by cells undergoing chondrogenic differentiation stained with hematoxylin and/or eosin (left) and with Masson trichrome (right) (A) 20 and (B) 40.

3.2. Checking for the presence of pericytes 3.2.1. Osteogenic differentiation After 10 d of administration of the treatments for osteogenic differentiation, the cultured cells did not show a clear differentiation to this cell type, although they showed a tendency to form the characteristic agglomerates (Fig. 7). 3.2.2. Adipogenic differentiation After 12 d of differentiative treatments cells were stained with Oil Red O methodology; while the control resulted negative, cells subjected to treatment showed the typical red color, which highlights the fat vacuoles of adipocytes (Fig. 8). 3.2.3. Chondrogenic differentiation After 10 d of differentiative treatments cells showed a tendency to aggregate and to detach from the coverslip, forming the characteristic clumps. To highlight cell morphology, these aggregate sections were stained with hematoxylin and/or eosin and Masson trichrome staining through which the cytoplasm appeared red, while the collagen, the main product of the chondrocytes, displayed a blue color (Fig. 9). 4. Discussion Angiogenesis is a process that, as a rule, does not take place physiologically in the adult, while it is mainly present in pathologic conditions, such as wound repair and development of the vascular network that supports the neoplastic progression.

An exception to the physiological quiescence of the vascular system is represented by the female reproductive system, where the formation of new blood vessels plays a key role, particularly during follicular development in the ovary and the subsequent formation of the corpus luteum, a transient endocrine organ in which the formation and regression of new vessels cyclically occur. Given that the corpus luteum represents a site of physiological angiogenesis, where endothelial cells play a main role in neovascularization, in the present work we isolated and characterized luteal endothelial cells. Their identity was confirmed by the characteristic “cobblestone” shape, typical of endothelial cells [35]. They grow as adherent cells showing contact inhibition and maintaining their morphology in long-term cultures [36]; moreover, they form the characteristic colony forming units, which are composed by a group of cells which, as they grow, “support” each other creating a larger colony [37]. The expression of CD31, CD105, VE-cad, Cav-1, vWF, VEGF, Flt-1, and KDR was demonstrated by RT-PCR. These markers are involved in important functions, particularly, in neovessel formation: CD31 is important in vascular remodeling [17], CD105 regulates the expression and activity of eNOS [18], vWF is essential in the interaction with platelets, VE-cad plays a pivotal role in the spatial organization of new blood vessels [17], Cav-1 interacts with the adhesion molecules and receptors signaling [10], VEGF and its receptors play an important role in the development and maintenance of the CL [38]. Isolated cells also displayed ability to produce the most important mediators of angiogenesis, NO, and VEGF [39]. Our data confirm that VEGF plays a role in endothelial cell migration [40] a process which is essential for angiogenesis and occurs under the guidance of chemotactic stimuli,

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involving the degradation of the extracellular matrix to facilitate the progression of migrating cells. The behavior of isolated cells under hypoxic condition is another aspect studied in this work. In fact, while oxygen shortage has been documented to play a crucial role in bovine CL development [41,42], the involvement of hypoxia in the regulation of CL function in the pig needs further supporting evidence [43]. Our data show, as documented by Liu et al [44], that vascular endothelial cell response to hypoxia is the increased expression and production of VEGF, by means of largely unexplored signaling mechanisms. Evidence suggests that ROS, including NO, may play a role in signal transduction during hypoxia [45] because their production by the mitochondrial complex III is critical for signaling in this condition and inhibitors of complex I abolish the induction of HIF-1a by hypoxia [46]. ROS increased levels in conditions of low O2 appear paradoxical but it must be taken into account that their mitochondrial production also varies according to the concentration of electron donors [47]. Therefore, the understanding of the mechanisms leading to ROS production during hypoxia and downstream effectors of the ROS hypoxic signaling should be better investigated [48]. Specifically, in this work we observed an inhibitory effect on NO production; while an increase of expression of NO synthase, the enzyme responsible for the production of this gas has been documented in cardiac myocytes and endothelial cells [49], transformed murine embryonic cells [50], and in primary endothelial cells [51]. Our data confirm the results of Fish et al [52] about a rapid decrease in the transcription of the gene eNOS/NOS3 in HUVEC induced by hypoxia. However, it is worth noticing that the role played by NO in the angiogenic process is not yet unequivocally clarified. The concentration reached by the molecule seems crucial: lower levels appear to activate HIF-1, stimulating the expression of VEGF, while high levels appear to inhibit the synthesis of VEGF [53]. Future studies aiming to study the role of ROS in the hypoxic response are needed to better clarify their involvement in regulating neovessel formation within the corpus luteum. The ability to form capillary-like structures in a 3dimensional matrix [26] is another significant behavior that we could highlight in isolated cells; in particular, cells adhering to microbeads of dextran [54] placed in a fibrin matrix, grow and organize themselves to form more complex structures. Therefore, on the basis of the overall data obtained, we conclude that the cells obtained by the described method can be useful to study the biology of angiogenesis. This information would improve the knowledge of a process that is often associated with diseases such as chronic arthritis, atherosclerosis, diabetic retinopathy, and especially tumors [55]. Recently, it has been suggested that pericytes contribute to maintain a subendothelial or perivascular niche, where resident cells are able to differentiate in mesenchymal stem cells (MSCs). Although a pericyte origin for MSCs is still debated, the presence of cells behaving as MSCs in niches of the vascular wall of microvessels is supported by different experimental finding [56] in different tissues where active angiogenesis occurs, as, for example, adipose tissue and endometrium [57,58].

Because corpus luteum is a site of active angiogenesis and is rich in microvessels, we have evaluated if our cell cultures comprise a population of cells able to exhibit a multidifferentiation potential comparable to MSCs toward osteogenic, adipogenic, and chondrogenic lineages. The obtained results show a positive chondrogenic and adipogenic differentiation while treatment for osteogenic differentiation has not been successful. Further investigations are needed to optimize the experimental protocols and to acquire other evidence confirming the presence of these cells. In conclusion, data collected in this study, demonstrating the ability to isolate endothelial cells from the corpus luteum, lead to suggest the ovary as a model for angiogenesis investigations aimed to better understanding the underlying molecular mechanisms: this aspect appears crucial for the development of therapeutic strategies against diseases in which this process is critical for their progression.

Acknowledgments This research was supported by grant of the Università degli Studi di Parma (FIL).

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