The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

Kata Osz
Michelle Ross
Jim Petrik
The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis
R E S E A R C H Open Access
Background: Ovarian angiogenesis is a complex process that is regulated by a balance between pro-and anti-angiogenic factors. Physiological processes within the ovary, such as folliculogenesis, ovulation, and luteal formation are dependent upon adequate vascularization and anything that disrupts normal angiogenic processes may result in ovarian dysfunction, and possibly infertility. The objective of this study was to evaluate the role of the thrombospondin-1 (TSP-1) receptor CD36 in mediating ovarian angiogenesis and regulating ovarian function. Methods: The role of CD36 was evaluated in granulosa cells in vitro and ovarian morphology and protein expression were determined in wild type and CD36 null mice.Results: In vitro, CD36 inhibition increased granulosa cell proliferation and decreased apoptosis. Granulosa cells in which CD36 was knocked down also exhibited an increase in expression of survival and angiogenic proteins. Ovaries from CD36 null mice were hypervascularized, with increased expression of pro-angiogenic vascular endothelial growth factor (VEGF) and its receptor VEGFR-2. Ovaries from CD36 null mice contained an increase in the numbers of pre-ovulatory follicles and decreased numbers of corpora lutea. CD36 null mice also had fewer number of offspring compared to wild type controls.Conclusions:The results from this study demonstrate that CD36 is integral to the regulation of ovarian angiogenesis by TSP-1 and the expression of these family members may be useful in the control of ovarian vascular disorders.

Background

Ovarian folliculogenesis, ovulation, and formation of the corpus luteum require complex and coordinated interaction of numerous autocrine, paracrine, and endocrine factors to regulate important physiologic processes such as angiogenesis. Angiogenesis is the formation of new blood vessels from pre-existing vasculature. In the adult, angiogenesis is generally quiescent except for the vascularization required for wound healing. However, the female reproductive tract, including the ovaries, and uterus are unique as they are adult tissues that undergo cyclic angiogenic processes to facilitate processes important to reproduction. An important regulator of ovarian angiogenesis is vascular endothelial growth factor (VEGF), which stimulates endothelial proliferation, migration, and survival, and we have shown that through interaction with its receptor VEGFR2, it is an important survival factor for extravascular ovarian cells [1].

Thrombospondin-1 (TSP-1) is a large (450 kDa) multimodular glycoprotein encoded by the THSB1 gene that was originally identified as a major component of platelet α-granules [2,3]. Since then, TSP-1 has been shown to be an important matricellular protein that mediates cell signaling, wound healing, cell adhesion, and angiogenesis [4,5]. Of TSP-1′s many biological functions, likely the most notable effect is the inhibition of angiogenesis [6]. The effects of TSP-1 in tumorigenesis have been well-studied. Reduced expression of TSP-1 is usually associated with aggressive angiogenesis and enhanced tumour formation [7], while TSP-1 over-expression generally results in inhibited tumour formation and reduced tumour vessel density [8]. During tumour development, TSP-1 is typically inversely associated with pro-angiogenic markers such as vascular endothelial growth factor (VEGF) where tumours with a higher TSP-1:VEGF ratio are typically slower growing, while tumours that favor VEGF expression are aggressive and have a higher rate of malignancy [9,10].

CD36 is a multi-ligand glycoprotein receptor, although it is best known for binding TSP-1. CD36 is known to act as a transporter and sensor of free fatty acids [11,12] and a scavenger receptor that binds a number of factors and participates in the internalization of cells, pathogens, and various lipoproteins [13]. CD36 is an 88-kDa integral plasma membrane protein and is expressed by a variety of cells including platelets, erythrocytes, endothelial cells, monocytes, granulosa, theca, and tumour cells [14][15][16]. Likely one of the most well-established functions of CD36 is the inhibition of angiogenesis following binding to the Type I repeats (TSR) of TSP-1 and −2 [17,18]. Following binding of TSR, CD36 initiates antiangiogenic signals through the induction of endothelial cell apoptosis [19,20]. TSP-1 stimulates CD36 to recruit non-receptor protein kinases fyn, lyn and yes to the CD36 complex [21]. This recruitment results in the activation of the kinase and initiation of the p38 mitogen activated protein kinase (MAPK) pathway, initiation of caspase-3-like effectors, and ultimately apoptotic cell death [14].

TSP-1 and CD36 are coordinately expressed in granulosa and theca cells of rodent [16] and bovine [15] ovaries. Expression of the ligand and receptor vary throughout the reproductive cycle and are regulated at least in part by gonadotropins [16]. Cyclical expression of TSP-1 is associated with changes in ovarian angiogenesis, where reduced expression of the protein accompanies increased perafollicular and luteal angiogenesis during period of follicular and luteal development [16].

We have shown that TSP-1 Null mice are subfertile and have altered ovarian morphology highlighted by increased vascularization and disrupted follicle dynamics, compared with wild-type controls [22]. TSP-1 and its mimetic peptides have been shown to reduce VEGF expression, inhibit ovarian angiogenesis, and induce follicle atresia [23,24]. These findings led us to believe that TSP-1 and pro-angiogenic VEGF had reciprocal inhibitory influences in the ovary and we demonstrated that TSP-1 bound VEGF, resulting in its internalization and degradation through the low density lipoprotein receptor related protein (LRP)-1. However, the extent of the ovarian morphological and functional alterations in the TSP-1 null mice could not be accounted for exclusively by a direct interaction between TSP-1 and VEGF. We anticipated that in addition to VEGF-mediated effects, CD36 signaling may have specific influences on ovarian function.

In ovarian tumor development, we demonstrated that treatment with TSP-1 mimetic peptides inhibited tumor formation, and that this effect was abrogated when CD36 expression was knocked down [25]. Based on the coordinated expression of TSP-1 and CD36 during ovarian follicle development, and the results from our work in the ovarian cancer model, we hypothesized that CD36 is an important mediator in the regulation of ovarian cell function and folliculogenesis.

Methods

Cell lines and tissue collection

Spontaneously immortalized rat granulosa cells (SIGC) were generously provided by Dr. Robert Burghardt (Texas A&M University, College Station, TX) and were cultured in DMEM/F12 (Gibco BRL), supplemented with 10% FBS, and 1% Antibiotic/Antimycotic (Gibco BRL). The SIGCs are derived from primary rat ovarian granulosa cell cultures and grow in culture without undergoing luteinization [26]. We have previously shown that these cells express the CD36 receptor [16]. CD36-null mice and wild type (WT) C57BL-6 littermates were a generous gift from Dr. Arend Bonen, Department of Health and Human Nutrition, University of Guelph, Guelph, ON. All animal work was carried out in compliance with guidelines established by the Canadian Council on Animal Care. For in vivo tissue collection, approximately 30-week old littermate WT and CD36 -\mice (n = 8/group) were injected intraperitoneally with 2.5 IU PMSG (Sigma) to initiate a synchronous follicular wave. 48 hr after injection, mice were euthanized via CO 2 asphyxiation and ovaries were collected at the late antral/preovulatory phase of the cycle and either flash frozen for protein collection, or fixed overnight in 10% neutral buffered formalin.

CD36 knockdown in SIGC

miRNA construct: The following complementary single stranded oligonucleotides were synthesized by Sigma-Aldrich: Rmi615640_top_cd36 -TGCTGTTCCTTGG CTAAATAACGAACGTTTTGGCCACTGACTGACGT TCGTTATAGCCAAGGAA.

Oligonucleotide strands were annealed and ligated into pcDNA 6.2-GW/EmGFP-miR vector (Life Technologies) then transformed into TOP10 cells (Life Technologies) according to the manufacturer's instructions. In addition to the CD36 knockdown oligonucleotides, scrambled sequence oligos with similar G/C content (Life Technologies) were also used as recommended.

Plasmids were purified by the Qiagen plasmid purification mini kit. Plasmids were sequenced (Laboratory Services, University of Guelph) to confirm the sequence identity of the oligo. 1 × 10 5 /ml SIGC cells were cultured in DMEM\F12 (Gibco) supplemented with 10% FBS without antibiotic on 24 well plates. At approximately 70% confluence medium was removed, cells were washed in PBS, and 500 ul/well 1xOPTI-MEM reduced serum medium (Gibco) was added. Transfection was achieved using Lipofectamine2000 (Invitrogen) according to the manufacturer instructions. Stable cell lines were established from single-cell dilution colonies, plated in 10 ug/ ml blasticidin (Invitrogen) for approximately 2 weeks to ensure elimination of any non-transfected cells. Stable cell line generation was confirmed with confocal microscopy to localize plasmid GFP within the SIGCs. To confirm knockdown, brightfield/darkfield immunofluorescence overlays with a phase-contrast inverted fluorescence microscope and Western blot analysis were performed on WT, Scrambled, and CD36KD SIGC in the log growth phase (Figure 1).

Hormone assays, ovarian morphometry, and fertility

At the late antral/ovulatory phase of the ovarian cycle, WT and CD36-\-mice were killed by CO 2 asphyxiation and blood was collected in a heparinized syringe via cardiac puncture and centrifuged at 1,000 × g for 10 min at 4°C. The plasma supernatant samples were collected and stored at −80°C until assayed. LH was assessed by using an ELISA kit from Endocrine Technologies (Newark, CA) (sensitivity, 0.5 ng/ml; intra-assay variation, 7%; inter-assay variation, 9.8%). FSH was measured by using an ELISA kit from Biocode-Hycel (sensitivity, 0.2 ng/ml; intra-assay variation, 4.7%; inter-assay variation, 8.5%). Ovaries were also collected, fixed in 10% neutral buffered formalin, processed, embedded in paraffin and 5 μm thick sections were cut throughout the entire tissue with a rotary microtome. For ovarian morphology assessment, every 5 th slide was subjected to Hematoxylin/Eosin staining and was used for counting of follicles and corpora lutea. Pre-antral/antral follicles and corpora lutea were counted in the 2 groups of mice (n = 6/group). As an index of fertility, ovarian synchronization was performed in a separate cohort of WT and CD36−/− null littermate mice (n = 6/group). Mice were injected with 2.5 IU PMSG, followed 48 hr later with 5 IU hCG (Sigma) to induce ovulation. Mice were immediately paired with proven male breeder mice and left together for one week before the male was removed. Mice were monitored for approximately 20 days following mating and the number of offspring from the female mice was counted.

Cell proliferation and apoptosis

To identify the role of CD36 in TSP-1 mediated granulosa cell proliferation and apoptosis, SIGC were subjected to RNAi knockdown as described above. WT and CD36 knockdown (CD36 KD) cells were plated in 24 well plate on glass coverslips. At 60% confluence, medium was changed to reduced serum (1% FBS) DMEM/F12 medium (Gibco) overnight. Cultures were treated with 0, 20, 50 or 100 ng/ml of the TSP-1 mimetic peptide ABT-898 for 24 hours. Following treatment, cells were rinsed in PBS and fixed for 1 hour in 10% (vol/vol) neutral buffered formalin. Cells were then permeabililzed with 1% Triton X-100 (Sigma) in PBS for 15 min, followed by blocking in 5% BSA/0.1% Sodium Azide in PBS for 10 min. Cells were then either incubated overnight at 4°C with antibodies to phosphorylated Histone H3 antibody (proliferation; 1:2000 dilution; Abcam, ab5176) or anti-active Caspase-3 antibody (apoptosis; 1:500 dilution; Millipore, ab3623) followed by Alexa-Fluore 594-labeled donkey anti-rabbit secondary antibody (1:500 dilution, Invitrogen) for 1 hr at room temperature. After rinsing, cells were stained with 2ug/ml DAPI (Sigma) to counterstain nuclei blue and mounted on glass slides (SuperFrost Plus, Fisher) with Prolong Gold antifade solution (Invitrogen). Epifluorescence microscopy was used for image acquisition and integrated morphometry software (Metamorph, Burlingname, CA) was used to quantify the percent immunopositive cells in follicles without (pre-antral) or with (antral) an antrum, and in corpora lutea.

Immunohistochemistry

Five micrometer-thick paraffin embedded ovarian tissue sections from wild-type and CD36 -\mice were incubated overnight at 4°C in a humidified chamber with rabbit polyclonal anti-VEGF antibody (1:600 dilution; Santa Cruz Biotechnology, CA, sc152), rabbit polyclonal anti-VEGFR-2 antibody (1:200 dilution; Cell Signaling, 2479); goat polyclonal anti-TSP-1 antibody (1:600 dilution; Santa Cruz, sc59887); mouse monoclonal anti-CD31 (1:500 (See figure on previous page.) Figure 1 Expression of survival factors and the effects of the TSP-1 mimetic peptide ABT-898 in vitro. A. siRNA inhibition reduced expression of CD36 in spontaneously immortalized rat granulosa cells (SIGC) in vitro, as observed using brightfield/darkfield immunofluorescence overlay and Western blotting. B. Western blotting revealed that CD36 knockdown cells had increased expression of survival factors (VEGF, pVEGFR2, Bcl-2, and pAkt, compared to WT SIGC, or those transfected with a scrambled RNAi sequence. * -denotes statistically different (p < 0.05) protein levels in CD36 knockdown cells compared to WT cells or those transfected with a scrambled sequence. C. ABT-898 significantly (p < 0.005) decreased SIGC proliferation and increased activated caspase-3 expression, while CD36 KD abrogated this effect. # -statistically different (p < 0.05) proliferation or apoptosis compared to the serum free (SF) treatment within that genotype; * statistically difference (p < 0.05) between WT and CD36 KD groups. dilution; Abcam; ab28634); or mouse monoclonal anti-Ki67 antibody (1:500 dilution; Sigma, Oakville, ON, SAB4501880). The following day, biotinylated secondary antibody (1:100 dilution, Sigma) was applied for 2 hr at room temperature (RT), followed by horseradish peroxidase (Extravidin, 1:50 dilution, Sigma) for 1 hr at RT. Antigen localization was provided with incubation in DAB solution (SigmaFast 3,3′-Diaminobenzidine tablets), and tissues were counterstained with Carazzi's Hematoxylin, dehydrated, cleared in xylene and mounted on coverslips. Images were captured using brightfield microscopy and the percentage of immunopositive tissue was quantified using a computer-generated thresholding algorithm and analysis (Aperio, ImageScope) for VEGF, VEGFR-2, and TSP-1 immunostaining. For CD31 staining, blood vessel density was calculated as the percentage of ovarian tissue comprised of CD31-postive endothelium using integrated morphometry software (Metamorph, CA). Ki67 staining in follicular and luteal cells were quantified manually by two independent observers blinded to whether the slides belonged to WT or CD36 −/− mice.

Western blot analysis

Total cellular proteins were isolated from wild type and transfected SIGC cells and ovarian tissue from WT and CD36 −/− mice using standard RIPA buffer containing protease inhibitor cocktail. Denatured 20ug and 40ug protein were loaded to 4-15% gradient PAGE gel (Mini protean TGX Gel, BioRad) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore), blocked (5% skim milk in TBST) for 1 hour at room temperature. For in vitro experiments, primary antibodies with disparate molecular weights were incubated on the pvdf membrane that was cut around the area of the predicted molecular weight. Membranes were incubated with VEGF (1:500, Santa Cruz), TSP-1 (1:200, santa cruz), Bcl-2 (1:500, Novus Biological), VEGFR2 (1:2000, Cell Signaling), phospho VEGFR-2 (1: 500, Cell Signaling), CD36 (1:400, BD Pharmingen), Akt (1:100, Cell Signaling), phosphoAkt (1:500, Cell Signaling) and B-actin (1:4000, Cell Signaling) overnight at 4C on a rocking platform. Blots were washed in TBS with 1% Tween 20 (TBST) and incubated in appropriate dilutions of secondary antibodies (anti-rabbit IgG-HRP; anti-mouse IgG-HRP, Cell Signaling). Reactive protein was detected with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer) on X-ray film (Kodak ClinicSelect blue). For some blots in which one of the primary antibodies was similar size to β-actin, the membrane was stripped using Millipore Re-Blot Plus MildTM (Millipore) for 15 min at RT, followed by 2 washes of 5% skim milk in TBST before re-probing with β-actin primary antibody and anti-rabbit secondary antibody. Films were imaged and densitometry analysis was performed using an AlphaInnotech imaging station.

Statistical analysis

Three replicates of all data were performed and used to determine statistical significance. For immunohistochemistry experiments, a minimum of 5 fields of view at 200× magnification were used. Statistical analysis was performed using a one-way ANOVA, followed by Bonferonni's post-hoc test. P values are listed in the figure legends.

Results

Knockdown of CD36 causes increased granulosa cell proliferation and survival

To evaluate the role of CD36 in granulosa cell function, we inhibited expression of the receptor in granulosa cells using RNA interference. With the RNA interference, we were able to reduce SIGC expression of CD36 by approximately 75% compared to WT cells or those transfected with scrambled oligonucleotide sequence ( Figure 1A/B). Following knockdown, SIGCs had increased expression of the pro-angiogenic proteins VEGF and phosphorylated VEGFR2 ( Figure 1B). SIGCs also exhibited an increase in protein levels of the cytoprotective proto-oncogene Bcl-2 and the pro-proliferative and pro-survival phosphorylated Akt ( Figure 1B). In in vitro experiments the Thrombospondin-1 mimetic peptide ABT-898 caused a significant (p < 0.05) decrease in SIGC proliferation in WT cells, but there was no change in proliferation in SIGCs that had CD36 knocked down ( Figure 1C). Conversely, ABT-898 induced SIGC apoptosis in WT cells, but this effect was abrogated with CD36 knockdown ( Figure 1C).

Ovarian morphometry, gonadotropin production and number of offspring are altered in CD36-\-mice Serum samples from WT and CD36 -\mice were collected at the time of euthanasia and subjected to ELISA analysis for leutenizing hormone (LH) and follicle stimulating hormone (FSH). FSH levels were significantly (p < 0.05) higher in CD36 null mice compared to WT controls (Figure 2A), while LH levels in circulation were significantly (p < 0.05) reduced compared to controls (Figure 2A). When ovarian structures were tabulated, there was a significant (p < 0.01) increase in the number of total follicles (including pre-antral, antral, and pre-ovulatory follicles) in CD36-\-mice compared to WT controls ( Figure 2B). Conversely, CD36 -\mouse ovaries contained significantly (p < 0.05) fewer corpora lutea than WT mouse ovaries ( Figure 2B). The number of live offspring born from CD36 -\mice was significantly (p < 0.05) lower than those from WT mothers ( Figure 2C).

Ovaries from CD36 -\mice have higher expression of pro-angiogenic and pro-survival factors, compared to ovaries from WT controls Mice from both groups had their ovarian cycles synchronized and ovaries were collected at the late antral/pre-ovulatory phase of the cycle. Immunohistochemistry was performed to localize and quantify expression of pro-angiogenic and pro-survival VEGF and it's receptor VEGFR-2, as well as TSP-1, which is the glycoprotein that binds and activates CD36. Ovaries from CD36 -\mice had significantly (p < 0.05) higher expression of VEGF and VEGFR-2 (Figure 3), while levels of TSP-1 protein remained unchanged (Figure 3).

CD36 -\ovaries have increased proliferation and vascularity

Ovaries from WT and CD36 -\mice collected at the late antral/pre-ovulatory phase of the ovarian cycle were immunostained for Ki67 to quantify cell proliferation in follicular granulosa cells. Ovaries from CD36 −/− demonstrated a significant (p < 0.05) increase in granulosa cell proliferation in both pre-antral and antral-stage follicles ( Figure 4A). Cellular proliferation in corpora lutea was not statistically different between groups ( Figure 4A). Immunostaining with the endothelial cell marker CD31 was also performed to quantify changes in ovarian vascularity. Ovaries from CD36 -\mice had a significant (p < 0.01) increase in microvessel density compared to WT control mice ( Figure 4B).

Discussion

This paper demonstrates the role of CD36 in the TSP-1 mediated effects in ovarian folliculogenesis. Based on the results of this study, CD36 appears to be an important regulator of ovarian angiogenesis, and follicular and luteal development. Disruption of expression of CD36 significantly alters ovarian morphology and expression of factors related to ovarian cell proliferation, survival, and angiogenesis. CD36 is expressed in granulosa and cumulus cells of rodents, bovines [16] and humans [27,28], although the specific function of the receptor is not completely understood.

In vitro, CD36 TSP-1 receptors are known to co-localize with VEGF receptors on cell membranes, and evidence suggests that these receptors may directly associate with each other and regulate signaling activity. We showed that knockdown of CD36 in ovarian granulosa cells resulted in an increase in expression of phosphorylated VEGFR2. The three type I repeat region of TSP-1 has been shown to reduce VEGFR2 phosphorylation and inhibit VEGF signal transduction [29] and the association between CD36, β1 integrins, and TSP-1 is thought to be important in mediating this inhibition [30]. In the CD36 null mice, there was a significant increase in ovarian blood vessel density compared to WT controls. In these mice, there was an increase in ovarian expression of VEGF and VEGFR2 and the in vitro data in this paper suggests that removal of the inhibitory influence of CD36 would allow for enhanced phosphorylation of VEGFR2, resulting in an increase in peri-follicular and luteal angiogenesis.

Knockdown of CD36 has been shown to increase proliferation and expression of survival and angiogenic in endothelial and tumor cells [25]. In this study, CD36 Figure 2 Serum gonadotropin levels, ovarian morphology, and number of offspring in WT and CD36−/− mice. A. ELISA was used to quantify changes FSH and LH in serum collected from CD36 −/− and WT mice. CD36 −/− mice had significantly (p < 0.05) higher circulating FSH and lower LH levels compared to WT controls. B. Morphometric analysis was conducted on histological sections from CD36 −/− and WT mice. Ovaries collected from CD36 −/− mice had significantly (p < 0.05) higher number of follicles and lower number of corpora lutei compared to WT controls. C. The number of offspring born to CD36−/− mice were fewer (p < 0.05) than WT control mice. knockdown in vitro resulted in increased granulosa proliferation and survival, which was associated with increased granulosa cell expression of phosphorylated Akt. Akt phosphorylation is an important promoter of granulosa cell proliferation and viability, which are the driving forces behind follicle growth and maturation [31,32]. In vitro, the increased expression of phosphorylated Akt was associated with increased granulosa cell proliferation and decreased apoptosis. In vivo, CD36 null mice had increased granulosa cell proliferation and a greater number of growing follicles, compared to WT controls. Akt phosphorylation promotes follicle activation and survival [33] and inhibits follicle atresia [34,35]. Increased activity of the PI3K/Akt pathway in the absence of CD36 could also have contributed to the increased number of follicles present in the ovaries of the CD36 null mice. Thrombospondin signaling has been linked to follicle atresia previously [15,23,24,36] although the mechanisms have been unclear. This paper suggests that the PI3/Akt signaling pathway may be an important mediator of TSP-1's effects in the ovary.

Normal function of the ovary is dependent on the tightly regulated angiogenic mechanisms that facilitate folliculogenesis, luteogenesis, and dissemination of the steroid hormones generated within the ovary. Angiogenesis within the ovary is a balance between and expression of pro-and anti-angiogenic factors. We have shown that members of the VEGF family and members of the anti-angiogenic TSP-1 are coordinately expressed during the ovarian cycle and have profound impacts on the angiogenic processes that occur throughout the cycle [15,16]. Within the ovary, cytokine action causes a downregulation of CD36, which is necessary to facilitate the explosive angiogenesis that occurs during luteal formation [37]. We showed in this study Figure 3 Ovarian expression of VEGF, its receptor VEGFR-2, and TSP-1 in CD36−/− and WT mice. A. Ovaries from CD36−/− and WT mice were immunostained for VEGF, VEGFR-2 and TSP-1. B. Immunohistochemical analysis showed that ovaries from CD36−/− mice had higher (p < 0.05) percentage positive ovarian tissue for VEGF and VEGFR-2 compared to WT controls. The percentage of tissue immunopositive for TSP-1 was not different between groups. C. Western blot analysis was performed on tissue lysates from WT and CD36−/− mice. D. There was a significant (* -p < 0.05) increase in VEGF and VEGFR-2 protein in CD36−/− ovaries compared to WT. No changes were observed in TSP-1 protein levels between groups. that CD36 null mice had significantly higher ovarian microvessel density compared to wildtype controls. CD36 appears to be critical in the regulation of physiological and pathophysiological angiogenesis. CD36 mediated TSP-1 signaling maintains corneal vascularity and CD36 deficiency leads to age-related corneal neovascularization while activation of CD36 can induce regression of inflammatory corneal angiogenesis [38]. CD36 has been implicated in the regulation of tumor angiogenesis. TSP-1, and other proteins containing the thrombospondin type-1 repeats (TSR), are known to endogenously inhibit the angiogenesis that occurs to facilitate tumor growth. CD36 has been shown to be required for the anti-angiogenic activity of these proteins and is absence impairs their angiogenesis inhibition and tumor vascularity increases and tumor growth is accelerated [17,39].

The morphological changes in the ovaries in the CD36 null mice somewhat replicate those seen in the condition of polycystic ovarian syndrome (PCOS) in which there is an increased number of primary follicles which remain preovulatory and do not progress to ovulation and formation of the corpus luteum [40]. In our study, CD36 null mice had an increased number of preovualtory follicles, concomitant with fewer corpora lutei, compared to WT controls. Elevated blood vessel density, and increased VEGF signaling were seen in the ovaries of the CD36 null mice and these are hallmarks of the pathogenesis of PCOS [41,42]. It has been shown that by increasing TSP-1 signaling, ovarian hypervascularization can be reversed as a method to treat PCOS [43]. Interestingly, CD36 null mice had elevated serum FSH, with suppressed LH, compared to WT controls. In classic PCOS, the hyperandrogenism and elevated GnRH generally results in an increase in the LH/FSH ratio [44]. In our mice, the elevated FSH may have been responsible for the increased number of recruited, but unovulated follicles as FSH is known to stimulate follicle development [45,46] and protect against follicle atresia [47,48]. FSH is a potent activator of the PI3K/akt signaling pathway [49] and the elevated circulating FSH seen in the CD36 null mice may have contributed to the increased akt phosphorylation observed in the ovaries from these mice. Reduced circulating LH in the CD36 null mice may have reduced the ovulatory stimulus, resulting in the increased number of pre-ovulatory follicles and fewer corpora lutei seen in these mice. CD36 has also been implicated in the pathogenesis of PCOS due to its role in regulating metabolism. CD36 is expressed in tissues regulated to fatty acid metabolism, including adipocytes [50]. Women with PCOS often exhibit higher levels of visceral obesity [51] and elevated CD36 expression in adipose tissue is seen in women with PCOS [11], further implicating the receptor in the pathogenesis of this disease. Levels of soluble CD36 are elevated in PCOS patients, and they are associated with the altered insulin sensitivity seen in this disease [52]. The metabolic profile of the CD36 null mice was not evaluated in this study, but will be the subject of investigation in the Figure 4 Cell proliferation and blood vessel density in ovaries from CD36−/− and WT mice. A. Immunohistochemistry for Ki67 showed that pre-antral and antral follicles from CD36−/− mice had a significant (p < 0.05) increase in the percentage of proliferating granulosa cells than WT controls. B. CD36−/− ovaries had a significantly (p < 0.05) higher blood vessel density than WT ovaries. Tissue sections are shown at 200× magnification; insets are at 600× magnification.

future. Transcriptome analysis has been performed on follicular cells from PCOS patients and healthy women. Haouzi et al. [53] showed that cumulus cells surrounding the oocyte had differential expression of a number of growth factors and genes involved in steroid metabolism. Granulosa cell expression of genes involved in inflammation, metabolism, and coagulation has also been implicated in the pathogenesis of PCOS [54,55]. After review of the gene lists published in the various transcriptome papers, CD36 was not specifically mentioned, although the cellular process that it is involved in have been implicated. A closer evaluation and comparison of CD36 expression in samples from PCOS patients and healthy women may be warranted.

Conclusions

The results from this study suggest that TSP-1 and CD36 have important functions in the ovary and are potent regulators of follicular and luteal development, ovarian angiogenesis, and ovarian function. Disrupted expression of these proteins may be related to ovarian dysfunction and their roles in specific ovarian pathologies warrants further investigation.

Figure 1 (
1See legend on next page.)
http://www.rbej.com/content/12/1/21
Competing interestsThe authors declare that they have no competing interests.Authors' contributions JP initiated and designed the project, performed data analysis and wrote the manuscript. KO performed the in vitro CD36 knockdown experiments and MR performed the immunohistochemistry and Western blot analysis. All authors read and approved the final manuscript version.
Vascular endothelial growth factor and its receptor, Flk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle. J Greenaway, K Connor, H G Pedersen, B L Coomber, J Lamarre, J Petrik, Endocrinology. 145Greenaway J, Connor K, Pedersen HG, Coomber BL, LaMarre J, Petrik J: Vascular endothelial growth factor and its receptor, Flk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle. Endocrinology 2004, 145:2896-2905.

The functions of thrombospondin-1 and-2. J Lawler, Curr Opin Cell Biol. 12Lawler J: The functions of thrombospondin-1 and-2. Curr Opin Cell Biol 2000, 12:634-640.

Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Lawler, J Cell Mol Med. 6Lawler J: Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 2002, 6:1-12.

The thrombospondins. J C Adams, J Lawler, Int J Biochem Cell B. 36Adams JC, Lawler J: The thrombospondins. Int J Biochem Cell B 2004, 36:961-968.

The thrombospondins. Cold Spring Harbor perspectives in biology. J C Adams, J Lawler, 39712Adams JC, Lawler J: The thrombospondins. Cold Spring Harbor perspectives in biology 2011, 3:a009712.

The face of TSR revealed: an extracellular signaling domain is exposed. R L Silverstein, J Cell Biol. 159Silverstein RL: The face of TSR revealed: an extracellular signaling domain is exposed. J Cell Biol 2002, 159:203-206.

Tumor progression: the effects of thrombospondin-1 and −2. J Lawler, M Detmar, Int J Biochem Cell B. 36Lawler J, Detmar M: Tumor progression: the effects of thrombospondin-1 and −2. Int J Biochem Cell B 2004, 36:1038-1045.

Regulation of tumor angiogenesis by thrombospondin-1. B Ren, Yee Ko, J Lawler, R Khosravi-Far, Biochim Biophys Acta. 1765Ren B, Yee KO, Lawler J, Khosravi-Far R: Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 2006, 1765:178-188.

Plasma vascular endothelial growth factor dysregulation in defining aggressiveness of head and neck squamous cell carcinoma. B Kulapaditharom, V Boonkitticharoen, C Sritara, J Oncol. 687934Kulapaditharom B, Boonkitticharoen V, Sritara C: Plasma vascular endothelial growth factor dysregulation in defining aggressiveness of head and neck squamous cell carcinoma. J Oncol 2012, 2012:687934.

Serum VEGF levels and tissue activation of VEGFR2/ KDR receptors in patients with breast and gynecologic cancer. M I Koukourakis, V Limberis, I Tentes, E Kontomanolis, A Kortsaris, E Sivridis, A Giatromanolaki, Cytokine. 53Koukourakis MI, Limberis V, Tentes I, Kontomanolis E, Kortsaris A, Sivridis E, Giatromanolaki A: Serum VEGF levels and tissue activation of VEGFR2/ KDR receptors in patients with breast and gynecologic cancer. Cytokine 2011, 53:370-375.

Omental adipose tissue overexpression of fatty acid transporter CD36 and decreased expression of hormone-sensitive lipase in insulin-resistant women with polycystic ovary syndrome. K M Seow, Y L Tsai, J L Hwang, W Y Hsu, L T Ho, C C Juan, Hum Reprod. 24Seow KM, Tsai YL, Hwang JL, Hsu WY, Ho LT, Juan CC: Omental adipose tissue overexpression of fatty acid transporter CD36 and decreased expression of hormone-sensitive lipase in insulin-resistant women with polycystic ovary syndrome. Hum Reprod 1982-1988, 2009:24.

Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis. J Zhou, M Febbraio, T Wada, Y Zhai, R Kuruba, J He, J H Lee, S Khadem, S Ren, S Li, R L Silverstein, W Xie, Gastroenterology. 134Zhou J, Febbraio M, Wada T, Zhai Y, Kuruba R, He J, Lee JH, Khadem S, Ren S, Li S, Silverstein RL, Xie W: Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis. Gastroenterology 2008, 134:556-567.

CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. R L Silverstein, M Febbraio, Sci Signal. 23Silverstein RL, Febbraio M: CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal 2009, 2:re3.

CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. M Febbraio, D P Hajjar, R L Silverstein, J Clin Invest. 108Febbraio M, Hajjar DP, Silverstein RL: CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest 2001, 108:785-791.

Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development. J Greenaway, P A Gentry, J J Feige, J Lamarre, J J Petrik, Biol Reprod. 72Greenaway J, Gentry PA, Feige JJ, LaMarre J, Petrik JJ: Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development. Biol Reprod 2005, 72:1071-1078.

Expression and localization of thrombospondin-1 and −2 and their cell-surface receptor, CD36, during rat follicular development and formation of the corpus luteum. J J Petrik, P A Gentry, J J Feige, J Lamarre, Biol Reprod. 67Petrik JJ, Gentry PA, Feige JJ, LaMarre J: Expression and localization of thrombospondin-1 and −2 and their cell-surface receptor, CD36, during rat follicular development and formation of the corpus luteum. Biol Reprod 2002, 67:1522-1531.

Context dependent role of the CD36-thrombospondin-histidine-rich glycoprotein axis in tumor angiogenesis and growth. J S Hale, M Li, M Sinyuk, W Jahnen-Dechent, J D Lathia, R L Silverstein, PloS One. 740033Hale JS, Li M, Sinyuk M, Jahnen-Dechent W, Lathia JD, Silverstein RL: Context dependent role of the CD36-thrombospondin-histidine-rich glycoprotein axis in tumor angiogenesis and growth. PloS One 2012, 7:e40033.

CD36-TSP-HRGP interactions in the regulation of angiogenesis. R L Silverstein, M Febbraio, Curr Pharm Design. 13Silverstein RL, Febbraio M: CD36-TSP-HRGP interactions in the regulation of angiogenesis. Curr Pharm Design 2007, 13:3559-3567.

Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1. T A Rege, J StewartJr, B Dranka, E N Benveniste, R L Silverstein, C L Gladson, J Cell Physiol. 218Rege TA, Stewart J Jr, Dranka B, Benveniste EN, Silverstein RL, Gladson CL: Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1. J Cell Physiol 2009, 218:94-103.

Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. J S Isenberg, G Martin-Manso, J B Maxhimer, D D Roberts, Nat Rev Canc. 9Isenberg JS, Martin-Manso G, Maxhimer JB, Roberts DD: Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. Nat Rev Canc 2009, 9:182-194.

Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. B Jimenez, O V Volpert, S E Crawford, M Febbraio, R L Silverstein, N Bouck, Nat Med. 6Jimenez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N: Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 2000, 6:41-48.

Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1). J Greenaway, J Lawler, R Moorehead, P Bornstein, J Lamarre, J Petrik, J Cell Physiol. 210Greenaway J, Lawler J, Moorehead R, Bornstein P, Lamarre J, Petrik J: Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1). J Cell Physiol 2007, 210:807-818.

Thrombospondin-1 inhibits angiogenesis and promotes follicular atresia in a novel in vitro angiogenesis assay. S A Garside, C R Harlow, S G Hillier, H M Fraser, F H Thomas, Endocrinology. 151Garside SA, Harlow CR, Hillier SG, Fraser HM, Thomas FH: Thrombospondin-1 inhibits angiogenesis and promotes follicular atresia in a novel in vitro angiogenesis assay. Endocrinology 2010, 151:1280-1289.

A thrombospondin-mimetic peptide, ABT-898, suppresses angiogenesis and promotes follicular atresia in pre-and early-antral follicles in vivo. S A Garside, J Henkin, K D Morris, S M Norvell, F H Thomas, H M Fraser, Endocrinology. 151Garside SA, Henkin J, Morris KD, Norvell SM, Thomas FH, Fraser HM: A thrombospondin-mimetic peptide, ABT-898, suppresses angiogenesis and promotes follicular atresia in pre-and early-antral follicles in vivo. Endocrinology 2010, 151:5905-5915.

ABT-510 induces tumor cell apoptosis and inhibits ovarian tumor growth in an orthotopic, syngeneic model of epithelial ovarian cancer. J Greenaway, J Henkin, J Lawler, R Moorehead, J Petrik, Mol Cancer Ther. 8Greenaway J, Henkin J, Lawler J, Moorehead R, Petrik J: ABT-510 induces tumor cell apoptosis and inhibits ovarian tumor growth in an orthotopic, syngeneic model of epithelial ovarian cancer. Mol Cancer Ther 2009, 8:64-74.

Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation. L S Stein, G Stoica, R Tilley, R C Burghardt, Cancer Res. 51Stein LS, Stoica G, Tilley R, Burghardt RC: Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation. Cancer Res 1991, 51:696-706.

Obese women exhibit differences in ovarian metabolites, hormones, and gene expression compared with moderate-weight women. R L Robker, L K Akison, B D Bennett, P N Thrupp, L R Chura, D L Russell, M Lane, R J Norman, J Clin Endocr Metab. 94Robker RL, Akison LK, Bennett BD, Thrupp PN, Chura LR, Russell DL, Lane M, Norman RJ: Obese women exhibit differences in ovarian metabolites, hormones, and gene expression compared with moderate-weight women. J Clin Endocr Metab 2009, 94:1533-1540.

Granulosa cell subtypes vary in response to oxidized low-density lipoprotein as regards specific lipoprotein receptors and antioxidant enzyme activity. H Serke, J Bausenwein, J Hirrlinger, M Nowicki, C Vilser, P Jogschies, F A Hmeidan, V Blumenauer, K Spanel-Borowski, J Clin Endocr Metab. 95Serke H, Bausenwein J, Hirrlinger J, Nowicki M, Vilser C, Jogschies P, Hmeidan FA, Blumenauer V, Spanel-Borowski K: Granulosa cell subtypes vary in response to oxidized low-density lipoprotein as regards specific lipoprotein receptors and antioxidant enzyme activity. J Clin Endocr Metab 2010, 95:3480-3490.

Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level. X Zhang, S Kazerounian, M Duquette, C Perruzzi, J A Nagy, H F Dvorak, S Parangi, J Lawler, FASEB J. 23Zhang X, Kazerounian S, Duquette M, Perruzzi C, Nagy JA, Dvorak HF, Parangi S, Lawler J: Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level. FASEB J 2009, 23:3368-3376.

Identification of CD36 molecular features required for its in vitro angiostatic activity. L Primo, C Ferrandi, C Roca, S Marchio, L Di Blasio, M Alessio, F Bussolino, FASEB J. 19Primo L, Ferrandi C, Roca C, Marchio S, di Blasio L, Alessio M, Bussolino F: Identification of CD36 molecular features required for its in vitro angiostatic activity. FASEB J 2005, 19:1713-1715.

PTEN and Akt expression during growth of human ovarian follicles. M Goto, A Iwase, H Ando, S Kurotsuchi, T Harata, F Kikkawa, J Assist Reprod Gen. 24Goto M, Iwase A, Ando H, Kurotsuchi S, Harata T, Kikkawa F: PTEN and Akt expression during growth of human ovarian follicles. J Assist Reprod Gen 2007, 24:541-546.

Murine folliculogenesis in vitro is stage-specifically regulated by insulin via the Akt signaling pathway. P Zhang, H Chao, X Sun, L Li, Q Shi, W Shen, Histochem Cell Biol. 134Zhang P, Chao H, Sun X, Li L, Shi Q, Shen W: Murine folliculogenesis in vitro is stage-specifically regulated by insulin via the Akt signaling pathway. Histochem Cell Biol 2010, 134:75-82.

Staying alive: PI3K pathway promotes primordial follicle activation and survival in response to 3MC-induced ovotoxicity. A P Sobinoff, B Nixon, S D Roman, E A Mclaughlin, Toxicol Sci. 128Sobinoff AP, Nixon B, Roman SD, McLaughlin EA: Staying alive: PI3K pathway promotes primordial follicle activation and survival in response to 3MC-induced ovotoxicity. Toxicol Sci 2012, 128:258-271.

Gonadotropin and intra-ovarian signals regulating follicle development and atresia: the delicate balance between life and death. J Craig, M Orisaka, H Wang, S Orisaka, W Thompson, C Zhu, F Kotsuji, B K Tsang, Frontiers Biosci. 12Craig J, Orisaka M, Wang H, Orisaka S, Thompson W, Zhu C, Kotsuji F, Tsang BK: Gonadotropin and intra-ovarian signals regulating follicle develop- ment and atresia: the delicate balance between life and death. Frontiers Biosci 2007, 12:3628-3639.

Angiopoietin 1 reduces rat follicular atresia mediated by apoptosis through the PI3K/Akt pathway. F Parborell, D Abramovich, G Irusta, M Tesone, Mol Cell Endocrinol. 343Parborell F, Abramovich D, Irusta G, Tesone M: Angiopoietin 1 reduces rat follicular atresia mediated by apoptosis through the PI3K/Akt pathway. Mol Cell Endocrinol 2011, 343:79-87.

Thrombospondin-1 expression is increased during follicular atresia in the primate ovary. F H Thomas, H Wilson, A Silvestri, H M Fraser, Endocrinology. 149Thomas FH, Wilson H, Silvestri A, Fraser HM: Thrombospondin-1 expression is increased during follicular atresia in the primate ovary. Endocrinology 2008, 149:185-192.

Equine luteal function regulation may depend on the interaction between cytokines and vascular endothelial growth factor: an in vitro study. A Galvao, S Henriques, D Pestka, K Lukasik, D Skarzynski, L M Mateus, G M Ferreira-Dias, Biol Reprod. 86187Galvao A, Henriques S, Pestka D, Lukasik K, Skarzynski D, Mateus LM, Ferreira-Dias GM: Equine luteal function regulation may depend on the interaction between cytokines and vascular endothelial growth factor: an in vitro study. Biol Reprod 2012, 86:187.

Emerging roles for the CD36 scavenger receptor as a potential therapeutic target for corneal neovascularization. B R Mwaikambo, C Yang, H Ong, S Chemtob, P Hardy, Endoc Metab Immune Disord Drug Targets. 8Mwaikambo BR, Yang C, Ong H, Chemtob S, Hardy P: Emerging roles for the CD36 scavenger receptor as a potential therapeutic target for corneal neovascularization. Endoc Metab Immune Disord Drug Targets 2008, 8:255-272.

Van Meir EG: Vasculostatin inhibits intracranial glioma growth and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism. B Kaur, S M Cork, E M Sandberg, N S Devi, Z Zhang, P A Klenotic, M Febbraio, H Shim, H Mao, C Tucker-Burden, R L Silverstein, D J Brat, J J Olson, Cancer Res. 69Kaur B, Cork SM, Sandberg EM, Devi NS, Zhang Z, Klenotic PA, Febbraio M, Shim H, Mao H, Tucker-Burden C, Silverstein RL, Brat DJ, Olson JJ, Van Meir EG: Vasculostatin inhibits intracranial glioma growth and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism. Cancer Res 2009, 69:1212-1220.

Stockpiling of transitional and classic primary follicles in ovaries of women with polycystic ovary syndrome. G A Maciel, E C Baracat, J A Benda, S M Markham, K Hensinger, R J Chang, G F Erickson, J Clin Endocr Metab. 89Maciel GA, Baracat EC, Benda JA, Markham SM, Hensinger K, Chang RJ, Erickson GF: Stockpiling of transitional and classic primary follicles in ovaries of women with polycystic ovary syndrome. J Clin Endocr Metab 2004, 89:5321-5327.

Angiopoietins/TIE2 system and VEGF are involved in ovarian function in a DHEA rat model of polycystic ovary syndrome. D Abramovich, G Irusta, D Bas, N I Cataldi, F Parborell, M Tesone, Endocrinology. 153Abramovich D, Irusta G, Bas D, Cataldi NI, Parborell F, Tesone M: Angiopoietins/TIE2 system and VEGF are involved in ovarian function in a DHEA rat model of polycystic ovary syndrome. Endocrinology 2012, 153:3446-3456.

Vascular endothelial growth factor and basic fibroblast growth factor in polycystic ovary syndrome during controlled ovarian hyperstimulation. P G Artini, M Monti, C Matteucci, V Valentino, F Cristello, A R Genazzani, Gynecol Endocrinol. 22Artini PG, Monti M, Matteucci C, Valentino V, Cristello F, Genazzani AR: Vascular endothelial growth factor and basic fibroblast growth factor in polycystic ovary syndrome during controlled ovarian hyperstimulation. Gynecol Endocrinol 2006, 22:465-470.

Metformin decreases angiogenesis via NF-kappaB and Erk1/ 2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1. B K Tan, R Adya, J Chen, S Farhatullah, D Heutling, D Mitchell, H Lehnert, H S Randeva, Cardiovasc Res. 83Tan BK, Adya R, Chen J, Farhatullah S, Heutling D, Mitchell D, Lehnert H, Randeva HS: Metformin decreases angiogenesis via NF-kappaB and Erk1/ 2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1. Cardiovasc Res 2009, 83:566-574.

Changes in luteinizing hormone and insulin secretion in polycystic ovarian syndrome. A M Fulghesu, F Cucinelli, V Pavone, F Murgia, M Guido, A Caruso, S Mancuso, A Lanzone, Hum Reprod. 14Fulghesu AM, Cucinelli F, Pavone V, Murgia F, Guido M, Caruso A, Mancuso S, Lanzone A: Changes in luteinizing hormone and insulin secretion in polycystic ovarian syndrome. Hum Reprod 1999, 14:611-617.

Effects of short-term injection of gonadotrophins on ovarian follicle development in hypogonadal (hpg) mice. D M Halpin, H M Charlton, J Reprod Fertil. 82Halpin DM, Charlton HM: Effects of short-term injection of gonadotrophins on ovarian follicle development in hypogonadal (hpg) mice. J Reprod Fertil 1988, 82:393-400.

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth. J R Mcneilly, E A Watson, Y A White, A A Murray, N Spears, A S Mcneilly, Biol Reprod. 85McNeilly JR, Watson EA, White YA, Murray AA, Spears N, McNeilly AS: Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth. Biol Reprod 2011, 85:584-593.

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles. P Lin, R Rui, Mol Reprod Dev. 77Lin P, Rui R: Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles. Mol Reprod Dev 2010, 77:670-678.

Folliclestimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis. X L Wang, Y Wu, L B Tan, Z Tian, J H Liu, D S Zhu, S M Zeng, J Biol Chem. 287Wang XL, Wu Y, Tan LB, Tian Z, Liu JH, Zhu DS, Zeng SM: Follicle- stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis. J Biol Chem 2012, 287:10166-10177.

PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells. M E Hunzicker-Dunn, B Lopez-Biladeau, N C Law, S E Fiedler, D W Carr, E T Maizels, Proc Natl Acad Sci. 109Hunzicker-Dunn ME, Lopez-Biladeau B, Law NC, Fiedler SE, Carr DW, Maizels ET: PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells. Proc Natl Acad Sci U S A 2012, 109:E2979--2988.

CD36 level and trafficking are determinants of lipolysis in adipocytes. D Zhou, D Samovski, A L Okunade, P D Stahl, N A Abumrad, X Su, FASEB J. 26Zhou D, Samovski D, Okunade AL, Stahl PD, Abumrad NA, Su X: CD36 level and trafficking are determinants of lipolysis in adipocytes. FASEB J 2012, 26:4733-4742.

Circulating and cellular adiponectin in polycystic ovary syndrome: relationship to glucose tolerance and insulin action. V Aroda, T P Ciaraldi, S A Chang, M H Dahan, R J Chang, R R Henry, Fertil Steril. 89Aroda V, Ciaraldi TP, Chang SA, Dahan MH, Chang RJ, Henry RR: Circulating and cellular adiponectin in polycystic ovary syndrome: relationship to glucose tolerance and insulin action. Fertil Steril 2008, 89:1200-1208.

Soluble CD36 and risk markers of insulin resistance and atherosclerosis are elevated in polycystic ovary syndrome and significantly reduced during pioglitazone treatment. D Glintborg, K Hojlund, M Andersen, J E Henriksen, H Beck-Nielsen, A Handberg, Diabetes Care. 31Glintborg D, Hojlund K, Andersen M, Henriksen JE, Beck-Nielsen H, Handberg A: Soluble CD36 and risk markers of insulin resistance and atherosclerosis are elevated in polycystic ovary syndrome and significantly reduced during pioglitazone treatment. Diabetes Care 2008, 31:328-334.

Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome. D Haouzi, S Assou, C Monzo, C Vincens, H Dechaud, S Hamamah, Hum Reprod. 27Haouzi D, Assou S, Monzo C, Vincens C, Dechaud H, Hamamah S: Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome. Hum Reprod 2012, 27:3523-3530.

Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis. S Kaur, K J Archer, M G Devi, A Kriplani, J F Strauss, R Singh, J Clin Endocr Metab. 97Kaur S, Archer KJ, Devi MG, Kriplani A, Strauss JF 3rd, Singh R: Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis. J Clin Endocr Metab 2012, 97:E2016-E2021.

Differential expression of inflammation-related genes in the ovarian stroma and granulosa cells of PCOS women. J Schmidt, B Weijdegard, A L Mikkelsen, S Lindenberg, L Nilsson, M Brannstrom, 10.1186/1477-7827-12-21Mol Hum Reprod. 20Schmidt J, Weijdegard B, Mikkelsen AL, Lindenberg S, Nilsson L, Brannstrom M: Differential expression of inflammation-related genes in the ovarian stroma and granulosa cells of PCOS women. Mol Hum Reprod 2013, 20:49-58. doi:10.1186/1477-7827-12-21

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Osz, Reproductive Biology and Endocrinology. 1221The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Osz et al.: The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis. Reproductive Biology and Endocrinology 2014 12:21. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Daylight saving time, circadian rhythms, and cardiovascular health
2018

Roberto Manfredini
Fabio Fabbian
Rosaria Cappadona
Pietro Amedeo Modesti
Daylight saving time, circadian rhythms, and cardiovascular health

(0123456789) 1 3 Internal and Emergency Medicine
13201810.1007/s11739-018-1900-4Received: 24 April 2018 / Accepted: 17 June 2018 / Published online: 3 July 2018POINT OF VIEWDaylight saving time · Circadian rhythms · Chronobiology · Myocardial infarction · Gender · Sleep deprivation · Chronotype · Climate
Very recently, the European Parliament, called to decide on possible abolition of the Daylight Saving Time (DST), approved a resolution calling the scientific community to conduct a more in-depth evaluation. The question is based on disruption of body's circadian rhythms. We review here the relationship between DST and cardiovascular health. The available evidence suggests the existence of an association between DST and a modest increase of occurrence of acute myocardial infarction, especially in the first week after the spring shift. Possible mechanisms include sleep deprivation, circadian misalignment and environmental conditions. The role of gender and individual preference in circadian rhythms (chronotype) will need further assessment.

Background

For many years, clocks in the countries of the European Union (EU) have been put forward by 1 h on the last weekend of March, to achieve the daylight saving time (DST). Several months ago, Finland called for the abolition of DST across the EU based on concerns for human health. The European parliament approved a resolution (384 votes in favour, 153 against, 12 abstentions) calling for the European commission and the scientific community to conduct a morein-depth "full evaluation."

Changing the time disrupts our body clocks. For most people, changing the time-even if only by 1 h-may produce tiredness as a small inconvenience. Other people, however, can have more serious consequences. The complex interaction among circadian body clocks and health represents a broad topic, explored by a growing body of literature, which cannot be reviewed in this study. We aim to provide information on circadian rhythms, their regulation and disruption, and possible effects on the cardiovascular (CV) system.

Circadian rhythms

All living organisms are characterized by multiple mechanisms to favour the synchronization and adaptation to the environment and its variations, and rhythmic organization represents a pivotal mechanism. Biological rhythms exist at any level of living organisms and, according to their cycle length, may be classified by Latin terms into the following: (a) circadian rhythms (from 'circa-diem': period of approximately 24 h), (b) ultradian rhythms ('ultra-diem': period shorter than 24 h, e.g., hours, minutes, and seconds), and (c) infradian rhythms ('infra-diem': period longer than 24 h, e.g., days, weeks, and months) [1].

3

Circadian rhythms, the most widely studied, are driven by either central or peripheral clocks. The central circadian clock (called 'master clock') is located within the suprachiasmatic nucleus (SCN) of the hypothalamus and accounts for approximately 20,000 neurons. Circadian clocks roughly consist of a set of proteins, capable of generating self-sustained positive and negative transcriptional feedback loops with a free-running period of approximately 24 h. However, peripheral circadian clocks have also been found within the great majority of mammalian cells and tissues. The central clock and the peripheral clocks can directly and indirectly influence numerous functions, including sleep/rest and locomotor activities, feeding and drinking behaviour, core body temperature, endocrine activity, metabolism, autonomic and sympathetic activity, and many others [2]. The CV system has biological clocks, as well. In fact, biological clocks have been found in the heart (at the cardiomyocyte level), blood vessels, and vascular endothelial cells. For example, the circadian clock within cardiomyocytes directly regulates myocardial metabolic gene expression, most likely in anticipation of the sleep/wake and feeding/fasting cycle. Thus, the results obtained on murine models show that the circadian clock within the cardiomyocytes allows for the rapid adaptation of the heart to the prolongation of fasting. Thus, a desynchronization of an organism with its environment, through circadian clock derangements, may also lead to the development of CV diseases [2].

Disruption of circadian rhythms

Circadian clocks regulate metabolism in many systems and organs, including the heart, skeletal muscle, liver, and adipose tissue. In particular, the cardiomyocyte circadian clock is essential for the responsiveness of the heart to fatty acids. When the cell cannot face increased fatty acid availability due to its inability to adequately increase fatty acid utilization, an accumulation of detrimental intracellular long-chain fatty acid derivatives occurs. This accumulation, occurring via multiple mechanisms, may affect the contractile function of the heart, as well as a series of other components, such as glucose intolerance, insulin resistance and insufficiency, dyslipidaemia, and increased vascular resistance [2]. Conversely, the circadian clock within the heart is altered in various animal models of human disease, including hypertension, diabetes, myocardial infarction, and simulated shift work.

Shift work, a circadian misalignment resulting from a 12-h inversion of the behavioural cycle (including sleep/ wake and fasting/feeding cycles), is a typical example of rhythm disruption. Shift workers are exposed to a misalignment of their behavioural and environmental cycles, which is a risk factor for hypertension, inflammation, and CV disease. It has been observed that compared with circadian alignment, circadian misalignment can (a) increase both systolic and diastolic blood pressure (BP); (b) decrease heart rate (HR) during wake periods and increase HR during sleep time; (c) reduce the sleep opportunity-associated dip in BP and HR; (d) affect the 24-h urinary epinephrine and norepinephrine excretion rates; (e) decrease markers of cardiac vagal modulation; (f) increase inflammatory markers, particularly 24-h interleukin-6 (IL-6), 24-h C-reactive protein (CRP), resistin, and tumour necrosis factor (TNF); and (g) decrease plasminogen activator inhibitor-1 (PAI-1) levels. Taken together, all these effects can explain the increased prevalence of CV disease in night workers versus day workers [3].

Daylight saving time: an insidious form of disruption?

DST refers to the practice of adjusting the local clock time. In 1784, Benjamin Franklin first analysed the cost of candle consumption during dark evenings in Paris, but, only in 1916, DST was adopted in Great Britain due to the costs of energy during the war. Despite common opinion that the DST spring shift leads to the relatively inconsequential loss of 1 h of sleep on the same night, increased sleep fragmentation and sleep latency present a cumulative effect of sleep loss at least across the following week, possibly longer ( Fig. 1). However, the autumn transition is simply considered to be a gain of 1 h of sleep. Again, there is a little evidence of extra sleep on that night, and the cumulative effect of 5 consecutive days of earlier rise times following the autumn change suggests a net loss of sleep across the week. In any case, short sleepers are more disadvantaged in their efforts to adjust to the clock change in both spring and autumn [4].

Circadian clocks use daylight to entrain to the organism's environment. Entrainment is sufficiently exact that humans adjust to the east-west progression of dawn within a given time zone. Similar to other animals, humans are seasonal,  although seasonality in humans has drastically declined in industrialized countries over the last 60 years. DST might constitute an additional factor for the dissociation of human biology from the seasons [5]. However, since studies on the effects of DST transitions essentially investigate the potential re-entrainment of individuals to a new social schedule, it is important to consider the individual's circadian preference (chronotype), which can exhibit substantial variations within a given population. In fact, depending on genotype, gender, age, and light exposure, individual clocks will adopt a different phase relationship to dawn.

A preferred high-risk time for unfavourable cardiovascular events?

The CV system is organized according to a specific oscillatory order, and most CV functions exhibit circadian changes. Thus, circadian differences in the physiological status of the CV system give rise to rhythmic-and predictable-in-timevariations either in the susceptibility of human beings to morbid and mortal events, or in the ability to precipitate the overt expression of disease [1]. It is known that the occurrence of CV events exhibits peculiar temporal patterns that vary across 24 h of the day. These patterns coincide with the temporal variation in the (a) pathophysiological mechanisms that trigger CV events and (b) physiological changes in the body rhythms. A series of factors, not harmful if considered alone, are capable of triggering unfavourable events when they all occur within the same temporal window [1]. An evident circadian (morning) rhythm for acute myocardial infarction (AMI) has been understood for more than 2 decades. It has been estimated that the incidence rate of AMI onset is 40% higher in the morning period than throughout the rest of the day, and nearly 28% of morning AMIs (accounting for approximately 9% of all AMIs) are attributable to the morning excess [6]. Moreover, rupture or dissection of aortic aneurysms shows a circadian variation, with an evident morning preference, as well [7]. The similarity of temporal patterns of such different acute CV and cerebrovascular events suggests that they share common underlying mechanisms. Several pathophysiologic phenomena, such as increased BP, HR, sympathetic activity, basal vascular tone, vasoconstrictive hormones, increased viscosity, fibrinogen, platelet aggregability, and reduced fibrinolytic activity, exhibit prominent circadian rhythms with phases positively correlated with the timing of excess in CV events [7]. Moreover, the effect of stress on human cardiac, vascular, and haemostatic status appears to be circadian rhythmic, with the effects being greater in the morning [1]. An exaggerated CV response to behavioural challenges has been suggested as a potential factor that enhances the risk of hypertension and CV disease. Thus, individuals who exhibit large increases in BP during acute psychological stress are at risk of atherosclerosis, probably secondary to endothelial damage by a BP surge, via increased flow turbulence, and a subsequent release of inflammatory and pro-atherogenic cytokines. Emotional stress can also play a pivotal triggering role for ischaemic heart disease, as shown for the morning onset of Takotsubo syndrome [8].

Daylight saving time and acute myocardial infarction: does an association exist?

Janszky and Ljung first reported a higher incidence of AMI following the spring DST shift, which was more pronounced in women [9]. We reviewed the available literature on this association [10], and found another six studies, four conducted in Europe and two in the United States, accounting for a total of 87,994 cases. Although there were differences among each other, they all supported the existence of an association between DST and the risk of AMI, particularly after the spring DST transition, with an increase ranging from 4 to 29%. In particular, three studies report a higher incidence on Monday, and only four provide an analysis by separating subgroups by gender [10].

Sleep deprivation

The central master clock is primarily entrained by light, and reduced exposure to light during the day and over exposure to light at night due to artificial lighting may impair the circadian organization of sleep. Changes in sleep architecture during sleep disruption may lead to increased energy intake, reduced energy expenditure, and insulin resistance. Short (< 6 h) sleep has been associated with negative health outcomes including hypertension, diabetes mellitus, obesity, and even mortality. A recent study has shown that short sleep, compared with normal sleep, is associated with a significant increase in the relative risk (RR) of mortality due to all causes (RR 1.12). Moreover, short sleepers have increased rates of obesity (38%), diabetes mellitus (37%), coronary heart disease (26%), and hypertension (17%) [11].

Personal circadian preference (chronotype) and gender

In 1976, Horne and Ostberg published a milestone study on possible individual differences in circadian attitudes in the International Journal of Chronobiology. By means of a simple self-assessment questionnaire (Morningness-Eveningness Questionnaire, MEQ), they evaluated individual differences in the circadian variation of oral temperature. Subjects categorized as morning types (M-type) show a significantly earlier peak time than evening ones (E-type) and have a higher daytime temperature. Intermediate types (I-type) have temperatures between those of the other groups. Recently, we collected the available evidence dealing with the association between chronotype, gender, and general health [12]. Individuals with the evening chronotype show sleep-related issues, e.g., later bedtime and wake-up and decreased sleep quality and quantity. Moreover, these people are prone to skipping breakfast, to have reduced physical activity, unhealthy dietary patterns, and other habits (smoking and alcohol), along with poor glycaemic control, metabolic syndrome, and diabetes. There are conflicting findings on whether men and women differ in their energy balance responses to sleep disruption, but women seem to be more susceptible. In particular, the female-evening association gives rise to several problems. Excessive daytime sleepiness and poor sleep quality are higher among females, and female predominance in the rate of depression is observed in subjects with a delayed sleep-wake schedule. Moreover, disturbances of the sleep-wake cycle in E-type females can also be increased by the presence of nightmares. Evening association may also be linked to being overweight, a feature that may more significantly impact the emotional health of adolescent females. Chronotype may also influence cognitive patterns and academic achievement. Evening association, in fact, can be a problem for morning attention at school, which is more evident in E-type girls compared to E-type boys [12].

Does the climate have a role in daylight saving time-linked CV risk?

The external influences of the environment (mainly climate) on biological rhythms are commonly observed by physicians. In clinical practice, it is common to find patients with mild hypertension requiring pharmacological treatment only in the winter, while, in the summer, they can suspend or reduce drug intake. Public health providers are aware of the 5-30% excess of mortality experienced in winter by most countries, mainly due to cerebrovascular events, and frail elderly often perceive the end of winter as a triumphant passage of Cape Horn [13]. Likewise, following the pioneering observation of the Medical Research Council's trial [14], where seasonality is associated with larger BP variations than those induced by drugs, researchers have now found that a clinical trial should last at least 1 year to mitigate the bias introduced by seasonality. Finally, although epidemiologists are aware that the hypertension burden assessed in large surveys may provide different results by season, the importance of environmental variations on health sometimes remains neglected. It is a common opinion that the main consequences of the spring DST are mediated by changes in sleeping time.

To modulate this effect, the change in the local clock time is usually made on the night between Saturday and Sunday. Interestingly, as considered above, several studies report the highest incidence on Monday or at least in the first working days of the week [10]. One day (Sunday) is clearly insufficient to become accustomed to waking up an hour earlier in the morning. However, the phenomenon can be seen from a different point of view, and other actors could play a role in increasing the risk of AMI or stroke. During spring, a complex interaction exists between single variables such as environmental parameters (i.e., temperature or light exposure), behavioural factors (such as physical activity amplitude, type, and intensity), and DST initiation, and these single variables may mutually influence each other. Why should this complex interaction increase CV risk? Although lowering of temperature is commonly considered responsible for an increase in BP, the first risk factor for stroke, data from a population-based study including 45,787 ischaemic stroke patients in Tuscany (Italy) from 1997 to 2007, shows that every degree celsius increase in temperature difference over 24 h is associated with a significant 1.5% increase in ischaemic stroke admissions [15]. A simplistic reading of this relationship could suggest a direct role of increase, rather than decrease, temperature in stroke. However, the association is more complex. The marked increase of stroke hospitalizations observed in Tuscany when the ΔT850hPa exceeds 5 °C apparently is not observed with the significant increase of all strokes observed in the same study when the T850hPa decreases [15]. This apparent contradiction is solved knowing that most of the ΔT850hPa increases exceeding 5 °C (94% of days) occur during the coldest months of the year (October-March) and never during warmer months (June-September). This finding means that stroke increases when a substantial increase of ΔT850hPa starts from a relatively low daily average temperature value, typical of the coldest months of the year. Furthermore, all studies refer to air temperature measured outdoors, while subjects live in heated houses. These day-to-day temperature changes may thus be associated with behavioural changes that might, in turn, influence the risk of stroke hospitalizations. In spring, when the temperature rises, people spend more time outdoors, and those at risk can suffer the greatest consequences.

Conclusions

The results of this study suggest an association between DST and a modest increase of AMI occurrence, even if only after the spring shift. Furthermore, in contrast to earlier findings, a higher risk is not confirmed for women. On one hand, it is possible that, after the spring DST shift, even modest sleep deprivation and circadian misalignment may affect CV health, since it has been associated with 1 3 increases in sympathetic tone and catecholamine levels. On the other hand, it is known that most people adjust more readily to delays than to advances, i.e., they suffer less from jet lag after westward than after eastward flights. A similar pattern has been suggested for DST transitions. Moreover, adjustment to DST transitions is chronotype specific, and differences are more marked after the spring shift [5]. Thus, further studies should consider the assessment of individual chronotype, sleep characteristics, and gender. Unfortunately, less than 44% of recent studies on the time of onset of CV events provide separate analysis [16]. Finally, in some studies, Monday exhibits the highest frequency of AMI onset. This finding is not surprising, since Monday is a critical day for the onset of AMI, stroke, and Takotsubo cardiomyopathy [17][18][19], probably due to a series of unfavourable factors, including the stress of commencing weekly activities and higher catecholamine and BP levels.

Furthermore, mechanisms other than rhythm disruption may also play a role. In spring, when the temperature rises, people spend more time outdoors, and those at risk may suffer the greatest consequences. Therefore, it cannot be excluded that the excess CV risk observed on Monday after the DST spring shift may be attributable to the anticipated exposure to the outdoor environment. In this condition, the thermal perception of people might be especially worsened if subjects are surprised in the outdoor environment with inappropriate clothing. Correct clothing behaviour was, indeed, reported to contribute to preventing excess winter mortality [20]. The importance of clothing may be particularly relevant to low-income families and other economically vulnerable persons who are disproportionately exposed to cold temperatures. Waiting for further in-depth studies and decisions by the European lawmakers, and prior to drastically moving towards DST abolition, a smoother approach could be suggested. As we now, discourage males over 55 years old with hypertension, obesity, and metabolic syndrome to perform their daily jogging early in the morning, providing adequate information to the population, especially to the highestrisk individuals, could help. The following is an easy strategy: (a) move bedtime 1 h a few days prior to the spring shift, to limit sleep deprivation effects; and (b) take care in exposing oneself to abrupt changes of temperature in the immediate post-shift days. Could such an easy combination of sleep strategies, scarves, hats, and gloves effectively reduce the CV effects of DST?

Fig. 1
1Schematic representation of factors potentially contributing to a higher cardiovascular risk following the daylight saving time (DST) shift 1 3
AcknowledgementsWe are grateful to Dr. Claudia Righini, Dr. Daniele Bragatto, and the entire staff of the Biblioteca Interaziendale di Scienze della Salute, Azienda Ospedaliera-Universitaria 'S. Anna' of Ferrara, for their continuous support.Funding support This work was supported by a research grant "Fondo Ateneo Ricerca-FAR", University of Ferrara, Italy (prof. Roberto Manfredini).Compliance with ethical standardsConflict of interest The authors declare they have no conflicts of interest regarding the publication of this work.Statement of human and animal rightsThis article does not contain any studies with human participants or animals performed by any of the authors.Informed consent None.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Circadian rhythms and cardiovascular and other stress-related events. R Manfredini, B Boari, R Salmi, A M Malagoni, F Manfredini, Fink G (ed) Encyclopedia of stress. OxfordAcademic Press12nd ednManfredini R, Boari B, Salmi R, Malagoni AM, Manfredini F (2007) Circadian rhythms and cardiovascular and other stress-related events. In: Fink G (ed) Encyclopedia of stress, vol 1, 2nd edn. Aca- demic Press, Oxford, pp 500-505

Potential role for peripheral circadian clock dyssynchrony in the pathogenesis of cardiovascular dysfunction. M E Young, M S Bray, Sleep Med. 86Young ME, Bray MS (2007) Potential role for peripheral circadian clock dyssynchrony in the pathogenesis of cardiovascular dysfunc- tion. Sleep Med 8(6):656-667

Circadian misalignment increases cardiovascular disease risk factors in humans. C J Morris, T E Purvis, K Hu, Fajl Scheer, Proc Natl Acad Sci. 11310Morris CJ, Purvis TE, Hu K, Scheer FAJL (2016) Circadian mis- alignment increases cardiovascular disease risk factors in humans. Proc Natl Acad Sci USA 113(10):E1402-E1411

The impact of daylight saving time on sleep and related behaviours. Y Harrison, Sleep Med Rev. 174Harrison Y (2013) The impact of daylight saving time on sleep and related behaviours. Sleep Med Rev 17(4):285-292

The human circadian clock's seasonal adjustment is disrupted by daylight saving time. T Kantermann, M Juda, M Merrow, T Roennenberg, Curr Biol. 1722Kantermann T, Juda M, Merrow M, Roennenberg T (2007) The human circadian clock's seasonal adjustment is disrupted by daylight saving time. Curr Biol 17(22):1996-2000

Meta-analysis of the morning excess of acute myocardial infarction and sudden cardiac death. M C Cohen, K M Rohtla, C E Lavery, J E Muller, M A Mittleman, Am J Cardiol. 7911Cohen MC, Rohtla KM, Lavery CE, Muller JE, Mittleman MA (1997) Meta-analysis of the morning excess of acute myo- cardial infarction and sudden cardiac death. Am J Cardiol 79(11):1512-1516

Chronobiology in aortic diseases-'is this really a random phenomenon. R Manfredini, F Fabbian, F Manfredini, R Salmi, M Gallerani, E Bossone, Progr Cardiovasc Dis. 561Manfredini R, Fabbian F, Manfredini F, Salmi R, Gallerani M, Bossone E (2013) Chronobiology in aortic diseases-'is this really a random phenomenon?'. Progr Cardiovasc Dis 56(1):116-124

Chronobiological patterns of onset of Tako-Tsubo cardiomyopathy: a multicenter Italian study. R Citro, M Previtali, D Bovelli, O Vriz, C Astarita, M M Patella, J Am Coll Cardiol. 542Citro R, Previtali M, Bovelli D, Vriz O, Astarita C, Patella MM et al (2009) Chronobiological patterns of onset of Tako-Tsubo cardiomyopathy: a multicenter Italian study. J Am Coll Cardiol 54(2):180-181

Shift to and from daylight saving time and incidence of myocardial infarction. I Janszky, R Ljung, N Engl J Med. 35918Janszky I, Ljung R (2008) Shift to and from daylight sav- ing time and incidence of myocardial infarction. N Engl J Med 359(18):1966-1968

Daylight saving time and myocardial infarction: should we be worried?. R Manfredini, F Fabbian, De Giorgi, A Zucchi, B Cappadona, R Signani, F , Eur Rev Med Pharmacol Sci. 223Manfredini R, Fabbian F, De Giorgi A, Zucchi B, Cappadona R, Sig- nani F et al (2018) Daylight saving time and myocardial infarction: should we be worried? Eur Rev Med Pharmacol Sci 22(3):750-755

Short sleep duration and health outcomes: a systematic review, meta-analysis, and metaregression. O Itani, M Jike, N Watanabe, Y Kaneita, Sleep Med. 32Itani O, Jike M, Watanabe N, Kaneita Y (2017) Short sleep duration and health outcomes: a systematic review, meta-analysis, and meta- regression. Sleep Med 32:246-256

Chronotype, gender and general health. F Fabbian, B Zucchi, De Giorgi, A Tiseo, R Boari, B Salmi, R , Chronobiol Int. 337Fabbian F, Zucchi B, De Giorgi A, Tiseo R, Boari B, Salmi R et al (2016) Chronotype, gender and general health. Chronobiol Int 33(7):863-882

The hazard of rounding Cape Horn: is it changing?. P A Modesti, Heart. 100Modesti PA (2014) The hazard of rounding Cape Horn: is it chang- ing? Heart 100(19):1489-1490

Seasonal variation in arterial blood pressure. P J Brennan, G Greenberg, W E Miall, S G Thompson, Br Med J Clin Res Ed. 2856346Brennan PJ, Greenberg G, Miall WE, Thompson SG (1982) Sea- sonal variation in arterial blood pressure. Br Med J Clin Res Ed 285(6346):919-923

Innovative approaches helpful to enhance knowledge on weather-related stroke events over a wide geographical area and a large population. M Morabito, A Crisci, R Vallorani, P A Modesti, G F Gensini, S Orlandini, Stroke. 423Morabito M, Crisci A, Vallorani R, Modesti PA, Gensini GF, Orlan- dini S (2011) Innovative approaches helpful to enhance knowledge on weather-related stroke events over a wide geographical area and a large population. Stroke 42(3):593-600

Sex and circadian periodicity of cardiovascular diseases. Are women sufficiently represented in chronobiological studies?. R Manfredini, R Salmi, R Cappadona, F Signani, S Basili, N Katsiki, Heart Fail Clin. 134Manfredini R, Salmi R, Cappadona R, Signani F, Basili S, Katsiki N (2017) Sex and circadian periodicity of cardiovascular diseases. Are women sufficiently represented in chronobiological studies? Heart Fail Clin 13(4):719-738

Seasonal and weekly patterns of hospital admissions for nonfatal and fatal myocardial infarction. R Manfredini, F Manfredini, B Boari, E Bergami, Mari E Gamberini, S , Am J Emerg Med. 279Manfredini R, Manfredini F, Boari B, Bergami E, Mari E, Gam- berini S et al (2009) Seasonal and weekly patterns of hospital admissions for nonfatal and fatal myocardial infarction. Am J Emerg Med 27(9):1097-1103

Monday preference in onset of takotsubo cardiomyopathy. R Manfredini, R Citro, M Previtali, O Vriz, Q Ciampi, M Pascotto, Am J Emerg Med. 286Manfredini R, Citro R, Previtali M, Vriz O, Ciampi Q, Pascotto M et al (2010) Monday preference in onset of takotsubo cardiomyopa- thy. Am J Emerg Med 28(6):715-719

Monday preference in onset of ischemic stroke. R Manfredini, I Casetta, E Paolini, O La Cecilia, B Boari, E Fallica, Am J Med. 1115Manfredini R, Casetta I, Paolini E, la Cecilia O, Boari B, Fallica E et al (2001) Monday preference in onset of ischemic stroke. Am J Med 111(5):401-403

Cold exposure and winter mortality from ischaemic heart disease, cerebrovascular disease, respiratory disease, and all causes in warm and cold regions of Europe. Lancet. 3499062The Eurowinter GroupThe Eurowinter Group (1997) Cold exposure and winter mortality from ischaemic heart disease, cerebrovascular disease, respira- tory disease, and all causes in warm and cold regions of Europe. Lancet 349(9062):1341-1346