女性保护血管的内分泌激素松弛素

© 2008 Bani, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. Vascular Health and Risk Management 2008:4(3) 515–524 515 R E V I E W Relaxin as a natural agent for vascular health Daniele Bani Department of Anatomy, Histology and Forensic Medicine, Sect. Histology, University of Florence, Italy Correspondence: Daniele Bani Viale G Pieraccini 6. I-50139, Florence Italy Tel +39 05 5427 1390 Fax +39 05 5427 1385 Email daniele.bani@unifi .it Abstract: Hypertension, atherothrombosis, myocardial infarction, stroke, peripheral vascular disease, and renal failure are the main manifestations of cardiovascular disease (CVD), the leading cause of death and disability in developed countries. Continuing insight into the patho- physiology of CVD can allow identifi cation of effective therapeutic strategies to reduce the occurrence of death and/or severe disabilities. In this context, a healthy endothelium is deemed crucial to proper functioning and maintenance of anatomical integrity of the vascular system in many organs. Of note, epidemiologic studies indicate that the incidence of CVD in women is very low until menopause and increases sharply thereafter. The loss of protection against CVD in post-menopausal women has been chiefl y attributed to ovarian steroid defi ciency. However, besides steroids, the ovary also produces the peptide hormone relaxin (RLX), which provides potent vasoactive effects which render it the most likely candidate as the elusive physiologi- cal shield against CVD in fertile women. In particular, RLX has a specifi c relaxant effect on peripheral and coronary vasculature, exerted by the stimulation of endogenous nitric oxide (NO) generation by cells of the vascular wall, and can induce angiogenesis. Moreover, RLX inhibits the activation of infl ammatory leukocytes and platelets, which play a key role in CVD. Experimental studies performed in vascular and blood cell in vitro and in animal models of vascular dysfunction, as well as pioneer clinical observations, have provided evidence that RLX can prevent and/or improve CVD, thus offering background to clinical trials aimed at exploring the broad therapeutic potential of human recombinant RLX as a new cardiovascular drug. Keywords: relaxin, blood vessels, endothelial cells, vascular smooth muscle, nitric oxide, cardiovascular disease The endothelium as a key player in vascular health and disease Hypertension, atherothrombosis, myocardial infarction, stroke, peripheral vascular disease, and renal failure are the main manifestations of cardiovascular disease (CVD), which is the leading cause of death and disability in most developed countries, also due to the progressive increase in people’s life span and expectancy (Wey 1992). Therefore, action should be taken to prevent CVD before its signs and symptoms appear or severe outcome, such as myocardial infarction or stroke, is experienced. In this context, there is a general agreement between basic and clinical scientists that a healthy endothelium is crucial to allow proper functioning and maintenance of anatomi- cal integrity of the vascular system in many organs, including the heart, brain, kidney, lung, and placenta. Conversely, damaged or dysfunctional endothelium substantially contributes to the pathogenesis and complications of CVD (Mensah 2007; Osto et al 2007). Public health interventions for health promotion and prevention of CVD and its associated risk factors often target the endothelium, and further understanding of endothelial function can help develop innovative approaches. Once viewed as a mere cell lining of the blood vessels serving as a barrier between the blood and the tissue extracellular matrix, the endothelium has been more recently recognized to be far more complex and functional (Figure 1). In fact, endothelial Vascular Health and Risk Management 2008:4(3)516 Bani cells play a major role in the regulation of vascular tone, hemostatic/fi brinolytic balance, angiogenesis, and leuko- cyte traffi cking (Mensah 2007). Many of the functional properties of the endothelial cells are attributable to their unique ability to synthesize and release a variety of vasoac- tive mediators, including nitric oxide (NO), a gaseous free radical and key signalling molecule (Moncada et al 1991). Intact endothelial cells constitutively express NO synthase III, which continuously generates nanomolar NO amounts. In turn, endothelium-derived NO accounts for vascular smooth muscle cell relaxation and vasodilatation (Palmer et al 1987; Moncada et al 1991), angiogenic response (Ziche et al 1994), and anti-adhesive properties of the inner endothelial surface to platelets (Radomski et al 1987) and leukocytes (Kubes et al 1991) by down-regulation of endothelial cell adhesion molecules (Sluiter et al 1993). Therefore, failure of endothelial NO pathway and increased oxidative stress with loss of the protective effect of NO tips the endothelial cell balance towards a pro-atherogenic and pro-thrombotic milieu (Nedeljkovic et al 2003) and can initiate a vicious cycle of events that result in vasomotor dysfunction and vascular infl ammation, which are essential components in the initiation and evolution of atherosclerosis (Napoli et al 2006). On these grounds, it is conceivable that agents that increase NO availability and reduce endothelial dysfunction may decrease susceptibility to CVD. A typical paradigm of the key importance of endothelial integrity for normal vascular function is represented by the pathogenic cascade initiated and sustained by cigarette smoke (Ambrose and Barua 2004). Smoking is known to increase the incidence of peripheral and coronary artery diseases, myo- cardial infarction and reduced foeto-placental perfusion and foetal growth restriction (Jonas et al 1992; Price et al 1999; Kalinka et al 2005; Zdravkovic et al 2005). Experimental and clinical investigations have provided evidence that cigarette smoke causes endothelial cell dysfunction, mainly consistent in decreased generation and bioavailability of NO, which results in defective endothelium-dependent vasodilatation and increased susceptibility to leukocyte and platelet adhesion (Barua et al 2001, 2002). Cigarette smoke is an example of the many dietary and lifestyle habits and inherited or acquired diseases predisposing to CVD, which also include obesity, hyperlipemia, diabetes, hypertension, vasculitis, and so on. In all these cases, the dysfunctional endothelium emerges as a crucial pathogenic factor and potential target for early diagnostic, preventative, and cura- tive strategies. Gender-related differences in CVD and the role of ovarian sex hormones Clinical and epidemiologic studies have shown that men and women suffering from CVD differ in pathophysiol- ogy, time for onset, and prognosis upon treatment. The incidence of CVD in women is very low until menopause and increases sharply thereafter to become similar to men, but with a 10–20-year delay (Wenger et al 1993; Tunstall et al 1994; Hu et al 2000). The epidemiologic fi ndings have been strengthened by the observation that, during fertile life, women have better endothelial function and vascular reactivity and higher NO biosynthesis than age-matched men (Forte et al 1998; Sader and Celermajer 2002). Female sex hormones have been obviously implicated in the protective effects of gender on the vasculature (Orshal and Khalil 2004; Mendelsohn and Karas 2005). Most of the existing literature Figure 1 The endothelial functions. Vascular Health and Risk Management 2008:4(3) 517 Relaxin and blood vessels is focused on ovarian steroids, estrogen and progesterone, which can infl uence vascular functions by multiple mecha- nisms. Estrogen and progesterone receptors are expressed by the vascular endothelium and smooth muscle (Khalil 2005): the interaction of ovarian steroids with cytosolic receptors triggers genomic effects leading to angiogenesis, endothelial NOS III up-regulation and smooth muscle growth inhibi- tion; on the other hand, stimulation of plasma membrane receptors by these hormones initiates non-genomic effects that stimulate endothelium-dependent vasorelaxation via NO-cGMP, prostacyclin-cAMP and hyper-polarization pathways (Hayashi et al 1995; Khalil 2005; Siow et al 2007). Moreover, ovarian steroids can also infl uence vascular func- tion by receptor-independent pathways, including direct antioxidant effects due to the presence of a phenolic group in their molecules (Siow et al 2007), as well as indirect effects mediated by hypothalamic-pituitary feed-back inhibition (Sader and Celermajer 2002). On the above grounds, the loss of protection against CVD in post-menopausal women has been chiefl y attributed to the defi ciency of ovarian steroids. Consequently, menopausal replacement therapy has been widely advocated for primary and secondary prevention (Grodstein et al 1996). However, estrogen or progesterone have not been clearly identifi ed as the protective agents in women. In fact, the results of estrogen/progestin replacement therapy are still controversial and under discussion (Herrington et al 2000; Rosano et al 2006). The conclusions drawn from randomized studies car- ried out by the Heart and Estrogen/progestin Replacement Study (HERS) Research Group (Hulley et al 1998) and the Women’s Health Initiative Investigators (WHI) (Manson et al 2003), enrolling large cohorts of subjects, concur to indicate that estrogen plus progestin does not confer cardiac protection and may even increase the risk of CVD among generally healthy post-menopausal women, especially during the fi rst year after the initiation of hormone use, most likely due to unfavorable effects of ovarian steroids upon platelet activation and coagulation (Bonnar 1987). Therefore, it has been recommended that estrogen/progestin replacement should not be prescribed for the prevention of cardiovascular disease (Manson et al 2003). This ostensible paradox can fi nd a logical explanation if bearing in mind that menopause causes the loss of ovarian function, which does not merely imply the cessation of estro- gen and progesterone production. In fact, besides steroids, the ovary produces the peptide hormone relaxin (RLX), which also has vasoactive properties. RLX is secreted by the corpus luteum and is secreted at detectable levels in the blood of cycling and pregnant women, being absent – or at least undetectable with the current assay methods – in post- menopausal women and in men (O’Byrne et al 1978; Eddie et al 1986; Khan-Dawood et al 1989; Stewart et al 1990; Winslow et al 1992). Could RLX be the elusive natural or physiological shield against CVD in fertile women? In the following chapters, the many points in favour of this hypoth- esis will be summarized and reviewed. RLX, its cognate molecules, and their receptors RLX was discovered in 1926 by Frederick Hisaw, who observed that the injection of serum from pregnant guinea pigs or rabbits into virgin guinea pigs induced signifi cant elongation of the interpubic ligament, attributed to stimula- tion of collagen turn-over (Hisaw 1926). Later on, he and his colleagues isolated from pregnant sow corpora lutea a peptide responsible for the observed relaxant effect on pubic symphisis and named it ‘relaxin’ (Fevold et al 1930). It is worth noting that the collagen remodeling properties of RLX fi rst highlighted by Hisaw have been confi rmed by many subsequent investigations (Unemori and Amento 1990; Unemori et al 1992, 1993, 1996; Garber et al 2001; Williams et al 2001; McDonald et al 2003; Samuel et al 2003) and remain as a hallmark of RLX and a paradigm of its specifi c biological effects (Sherwood 2004). RLX has been long included in the insulin hormone superfamily because of structural homologies with insulin: both peptides have similar 6 kDa molecular weights and are composed of A and B chains stabilized by inter- and intra- chain disulfi de bonds (Schwabe and Büllesbach 1994). More recently, however, it has been defi nitely ascertained that RLX is a member of a peptide hormone family that diverged from insulin early in vertebrate evolution; therefore, RLX has been assigned to a specifi c hormone family, termed the RLX peptide family, which includes three different RLXs, H1, H2 and H3, as well as insulin-like peptide (INSL)3, INSL4, INSL5 and INSL6 (Bathgate et al 2003; Samuel et al 2006). In humans, 3 separate RLX genes have been found and designated RLN1, RLN2 and RLN3 (Bathgate et al 2003; Samuel et al 2006). The peptide encoded by the RLN2 gene, H2 RLX, is the major circulating form and is produced mainly in the corpus luteum (Hudson et al 1984). Circulating RLX accounts for most of the known biological effects of the hormone in humans and experimental animals. RLX acts on the female reproductive system as well as on non-reproductive targets, including the cardiovascular system and the connective tissue (Bani 1997; Conrad and Vascular Health and Risk Management 2008:4(3)518 Bani Novak 2004; Sherwood 2004; Dschietzig et al 2006; Samuel et al 2006; Nistri et al 2007). RLX is the ligand for two leucine-rich repeat-containing G-protein coupled receptors (LGRs), LGR7 and LGR8 (Hsu et al 2002), now classifi ed as relaxin family peptide recep- tors 1 and 2 (RXFP1 and RXFP2), respectively (Bathgate et al 2006). RXFP1 is the main and most specifi c H2 RLX receptor, but it is also able to bind H1 and H3 RLX, although with less affi nity. On the other hand, RXFP2 chiefl y binds INSL3 and also H1 and H2 RLX, but with less affi nity (Hsu et al 2002; Sudo et al 2003). These receptors have been found on most if not all RLX target tissues and cells, and are abundantly expressed in the reproductive, nervous, renal and cardiovascular systems (Hsu et al 2002). More recently, other G protein-coupled receptors for peptides of the relaxin family have been discovered and termed RXFP3 (formerly GPCR135) and RXFP4 (formerly GPCR142) (Bathgate et al 2006). RXFP3 binds H3 RLX with high affi nity (Liu et al 2003a) while RXFP4 binds H3 RLX (Liu et al 2003b) and also INSL5 (Liu et al 2005). Moreover, RLX has been reported to act as an agonist for cytoplasmic glucocorticoid receptors, thereby exerting possible cortisol-like effects (Dschietzig et al 2004). Binding of RLX to its surface recep- tor results in the activation of multiple intracellular response pathways, which include cAMP and NO as second messen- gers (Hsu et al 2002; Nistri and Bani 2003). In particular, the observation that RLX can up-regulate NO biosynthesis in several of its targets underscores its potential as cardiovascu- lar hormone, NO being a key regulator of vascular function (Moncada et al 1991; Nathan 1992). RLX and the vascular system Peculiarly, the fi rst reports on the ability of RLX to infl uence blood vessels came from clinical rather than basic research. During the late 1950s and early 1960s, the Warner-Chilcott Laboratories provided an impure preparation of porcine RLX, commercially available as Releasin, and supported studies in humans that examined the use of relaxin as a therapeutic agent for scleroderma, cervical ripening and premature labor, based on its collagen-remodeling properties. Releasin, injected intramuscularly to patients suffering for peripheral vascular diseases and Raynaud’s syndrome, caused a dra- matic, albeit transient, amelioration of symptoms and signs of ischemia (Casten and Boucek 1958; Casten et al 1960). These effects were attributed to increased compliance of the blood vessels due to loosening of their adventitial extracel- lular matrix. However, Casten and colleagues also reported that, in some patients who also suffered for ischemic heart disease, Releasin treatment led to the reduction of the daily glyceryl trinitrate requirements. Retrospectively, this impor- tant observation clearly suggests that RLX does have a direct, dilatory effect on peripheral and coronary vasculature. Sub- sequent studies in monkeys by the Hisaw’s group described an enlargement of endometrial arterioles and capillaries (Dallenbach-Hellweg et al 1966) and provided fi rst evidence for an angiogenic effect of RLX on endothelial cells of the endometrial blood vessels (Hisaw 1967). Defi nite recogni- tion of the blood vessels as specifi c RLX targets came from the identifi cation of RLX binding sites/receptors on vascular cells in reproductive and non-reproductive organs (Min and Sherwood 1996; Kohsaka et al 1998; Hsu et al 2002). Vasodilatation may be regarded as an additional, specifi c hallmark of RLX, as it has been observed in many target organs and tissues, regardless of gender. RLX-induced vaso- dilatation mainly involves the distal segments of the vascular tree, namely arterioles provided with a smooth muscle coat as well as capillaries and post-capillary venules, which lack smooth muscle in their wall. Of note, RLX promotes vasodi- latation in reproductive organs, such as the uterus (Vasilenko et al 1986; Bani G et al 1995) and the mammary gland (Bani et al 1988), as well as in non-reproductive targets, including mesocaecum (Bigazzi et al 1986), kidney (Danielson et al 1999, 2000; Novak et al 2001), liver (Bani et al 2001), lung (Bani et al 1997), and heart (Bani Sacchi et al 1995; Masini et al 1997). Vasodilatation appears as a physiologic effect of RLX since it is fully manifest at hormone concentrations which are in the nanomolar range, similar to the RLX blood levels of normal human pregnancy (O’Byrne et al 1978; Eddie et al 1986). RLX is extremely potent as a vasorelax- ant: in the isolated, perfused rat and guinea pig heart, the dose-dependent increase in coronary fl ow induced by RLX is signifi cantly higher than that obtained with similar doses of typical vasodilatatory agents such as acetylcholine or sodium nitroprusside (Bani Sacchi et al 1995); in the mesocaecum, RLX counteracted similar concentrations of norepinephine (Bigazzi et al 1986). Vasodilatation also emerges as a pri- mary effect of RLX and is not secondary to tissue growth stimulation, as it could be appreciated even in the absence of any growth response. Of note, the vasodilatatory effects of RLX are most prominent in vasoconstricted blood vessels, rather than in normal blood vessels, suggesting that this hormone could act as a natural regulator of vascular tone and a shield against the derangements of vascular reactivity. In the rat kidney, RLX reduces the myogenic activity of small arteries and blunts the vasoconstrictive response to angiotensin II, in both genders Vascular Health and Risk Management 2008:4(3) 519 Relaxin and blood vessels (Danielson et al 1999, 2000; Novak et al 2001). In a rat model of renal artery ligation, RLX counteracts the increase in systemic blood pressure due to activation of the renin- angiotensin system (Garber et al 2003). In spontaneously hypertensive rats (SHR), but not normotensive rats, RLX reduces blood pressure and blunts the response of mesenteric vessels to vasoconstrictors (St Louis et al 1985). Besides vasodilatation, RLX can also induce angio- genesis in some target organs, as suggested by the pioneer fi ndings by Hisaw (1967), using partially purifi ed RLX. In human endometrial cells in vitro, RLX induces the produc- tion of potent angiogenic molecules such as basic fi broblast growth factor (bFGF) and vascular endothelial growth fac- tor (V