Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system

Abstract

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis.

Introduction

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily, which contains TGF-βs, BMPs, nodal, myostatin, activins/inhibins and anti-Mullerian hormone (Miyazono et al. 2005). Although initially identified as important signalling molecules in bone formation, BMPs are emerging as crucial players in the regulation of function in multiple tissues and organs (Wang et al. 2014), including the cardiovascular system (Cai et al. 2012). BMPs sequentially bind to type I and type II BMP receptors (Nohe et al. 2002), which are serine–threonine kinases, to transduce signals through both receptor-regulated Smad (R-Smad) and Smad-independent signalling pathways (Derynck et al. 2001; Shi & Massague, 2003). The specific type II receptor for BMPs is BMP receptor II (BMPRII). BMP type I receptors are also known as activin-receptor like kinases (ALKs), and ALK1, ALK2, ALK3 (BMPR1A) and ALK6 (BMPR1B) are the identified type I receptors bound by BMPs. Based on the binding preference of BMPs to type I receptors, BMPs can be classified into several subgroups (Miyazono et al. 2010). For example, BMP2/4 binds to ALK3 and ALK6; BMP5/6/7/8 interacts with ALK2 and ALK6; BMP9/10 exhibits a higher affinity for ALK1 and ALK2; while growth and differentiation factor (GDF) 5/6/7 preferentially activates ALK6. Since the BMP intracellular signal transduction mechanism has been reviewed elsewhere in relative detail (Lowery & de Caestecker, 2010; Chen et al. 2012), this article focuses more upon the regulation and consequences of BMP signalling in the cardiovascular system. In view of an increasing recognition of dysregulated BMP signalling in the pathogenesis of cardiovascular diseases, we aim to present recent advances in our understanding of the regulatory mechanisms of BMP signalling and its relevance to cardiovascular pathogenesis.

BMP signalling pathways

The control of BMP signal transduction can be achieved through manipulation of the signalling transducers (Fig.1). Hence, any molecules that regulate the expression or activity of these transducers can be considered as regulators of BMP signalling. Table1 presents a list of BMP–Smad signalling components, including ligands, receptors, Smad proteins and transcriptional cofactors, as well as summarizing typical regulators of BMP signalling.

Figure 1.

BMP–Smad signalling transducers and regulators

BMPs exert both paracrine and endocrine effects on target cells by activating type II and type I BMP receptors, which further signal to the major intracellular mediators, Smad proteins, to transcribe target gene expression. The general structures of Smad proteins are presented in the box. A number of BMP signalling regulators are illustrated, including BMP antagonists, small-molecule drugs, disturbed flow, pro-inflammatory stimuli, ubiquitin ligases and miRNAs.

Table 1.

BMP–Smad signalling components and regulators

	BMP2/4	BMP7	BMP9/10	Regulators for signalling transducers
Major	BMPRII	BMPRII	BMPRII	miR-17-5, miR-20a, miR-21, PKGI
Type II receptor				FK506, PPARγ agonist
Preferred	ALK3/6	ALK1/2	ALK1/2	Compound C, Smurf1/2, Smad6/7
Type I receptor
Co-receptor	RGMa	Endoglin	Endoglin	VEGF-Src, HO-1
Intracellular mediator	Smad1/5/8 Smad4			Smurf1/2, Smad6/7, MAPK, GSK3β, miR-26a
Transcriptional cofactor		p300,CBP, HDACs		TGIF
Regulators of local production of BMP2/4		Shear stress Statins NF-κB miR-141

BMP signalling components

BMP ligands

Among more than 20 ligands in the BMP family, the best-characterized BMPs in the cardiovascular system are BMP2, BMP4, BMP7 and BMP9. Both BMP2 and BMP4 are present in the circulating blood and they can be locally produced by various types of cells in the cardiovascular system (Dyer et al. 2014). BMP2 and BMP4 are pro-inflammatory and able to stimulate superoxide production and thus oxidative stress in cardiovascular cells (Csiszar et al. 2006; Wong et al. 2010; Sun et al. 2013a). They are implicated in the development of atherosclerosis due to their pro-inflammatory effects on endothelial cells (ECs) and the promotion of calcification of vascular smooth muscle cells (VSMCs) (Hayashi et al. 2006; Li et al. 2008). In sharp contrast to its harmful impact in the systemic circulation, BMP4 exerts an anti-inflammatory and anti-oxidant benefit on ECs of pulmonary arteries (Csiszar et al. 2008). Unlike BMP2 and BMP4, BMP7 was proposed to inhibit vascular calcification (Hruska et al. 2005). BMP7 is highly expressed in kidney and liver and is well known for its anti-fibrotic action in chronic renal injury (Zeisberg et al. 2003; Wang et al. 2006). However, the circulating source of BMP7 in vivo remains unclear. BMP9 was identified as a circulating vascular quiescence factor with a potent anti-angiogenic capacity in ECs (Scharpfenecker et al. 2007; David et al. 2008) although the circulating BMP9 is primarily derived from liver (Miller et al. 2000). The diverse roles of these BMPs in the pathogenesis of cardiovascular diseases are discussed below.

BMP receptors, co-receptors and decoy receptors

BMP receptors are widely expressed with a certain degree of cell type specificity. BMP ligands bind to two types of serine–threonine kinase receptors, namely type I and type II receptors, both of which are required for BMP signalling. Normally, BMPs first bind to type I receptors, which are phosphorylated by the type II receptors to trigger the BMP signalling cascade (von Bubnoff & Cho, 2001; Nohe et al. 2002). In the absence of type II receptors, BMPs can still bind to type I receptors but with lower affinity. The identified type II receptors for BMPs are BMPRII, activin receptor IIA (ActR-IIA) and activin receptor IIB (ActR-IIB). BMPRII is bound specifically by BMPs while ActRII and ActR-IIB are shared by activins, myostatins and BMPs (Yu et al. 2005). Type I receptors are commonly termed the activin receptor-like kinases (ALKs). ALK1, ALK2, ALK3 and ALK6 are the identified type I receptors for BMPs, with varied affinities. For example, BMP2 and BMP4 preferentially bind to ALK3 and ALK6, whereas BMP6 and BMP7 tend to interact strongly with ALK2 but weakly with ALK6. ALK1 expression is mostly restricted to ECs and a few other cell types (David et al. 2007) and ALK6 also exhibits a restricted expression profile, with greater expression in the brain, lung and ovary (Lee et al. 2009b). By contrast, ALK2 and ALK3 are more abundantly present in most cell types.

Although interaction between type II and type I receptors is usually sufficient to mediate BMP signal transduction, receptor binding and associated signalling activity in response to certain ligands can be also regulated by BMP co-receptors. Three types of BMP co-receptors have been identified and they are the repulsive guidance molecule (RGM) family, endoglin and betaglycan (TGFβR3). RGMs including RGMa, RGMb (Dragon) and RGMc (hemojuvelin) are co-receptors for BMP2 and BMP4 to enhance their signal transduction (Babitt et al. 2005, 2006; Samad et al. 2005), while RGMa can also facilitate BMP2 and BMP4 to utilize ActR-II as their type II receptor (Xia et al. 2007). Endoglin is a cell surface protein highly expressed in proliferating ECs, especially in tumour growth. Endoglin binds to BMP2/7 to inhibit TGF-β-induced cellular responses, while it promotes BMP7-stimulated activity, thus increasing ALK1–Smad1/5 signalling (Scherner et al. 2007). The release of soluble endoglin can be negatively regulated by haem oxygenase-1 in ECs (Cudmore et al. 2007). Additionally, epidermal growth factor and vascular endothelial growth factor (VEGF) as Src-activators were reported to negatively affect endoglin stability via Src-mediated phosphorylation of its juxtamembrane cytoplasmic tyrosine motif (Pan et al. 2014). Betaglycan, also known as TGFβ type III receptor, binds to several BMPs including BMP2, BMP4, BMP7 and GDF5 to augment their binding to type I receptors (Kirkbride et al. 2008).

Decoy receptors for BMPs negatively regulate BMP-triggered signalling by competing for the binding site of BMPs with type I receptors (Sieber et al. 2009). BMP and activin membrane-bound inhibitor (BAMBI) is a decoy receptor that resembles BMP type I receptor but does not contain an intracellular kinase domain, and thus it can sequester BMPs from active receptors (Onichtchouk et al. 1999). BAMBI was shown to protect murine heart from pressure-overload biomechanical stress through inhibiting TGF-β signalling (Villar et al. 2013).

Receptor-regulated Smads and inhibitory Smads

The major intracellular mediators of signalling initiated by the BMP ligands are Smad proteins (Derynck & Zhang, 2003). There are eight different Smad proteins in the TGF-β family. These Smads constitute three functionally distinct groups: receptor-regulated Smads (R-Smads, Smad1/2/3/5/8), common Smad (Smad4), and inhibitory Smads (I-Smads, Smad6/7). Smad1, Smad5 and Smad8 are activated by specific BMP type I receptor-mediated phosphorylation and interact with Smad4 to translocate into the nucleus, where they trigger gene transcription through binding to the Smad-binding elements (SBEs) in the gene promoter region (Kretzschmar et al. 1997b; Shi & Massague, 2003). On the other hand, Smad7 inhibits both TGF-β and BMP signalling while Smad6 is relatively specific for inhibiting BMP but not TGF-β signalling. Smad6 and Smad7 are also known as vascular mads because of their induction by laminar shear stress in ECs (Topper et al. 1997). Smad6 negatively regulates the BMP signalling by competing with Smad1 for type I receptor or Smad4 (Galvin et al. 2000).

The structural basis of different Smads determines their molecular behaviour. The R-Smads have two conserved domains separated by a linker region in the middle (Fig.1). The N-terminal MH1 domain is the DNA binding region while the C-terminal MH2 domain is an interaction domain for binding to other co-factors and nuclear translocation (Shi et al. 1997). There is an SSxS sequence in the C-terminal of R-Smads for phosphorylation by type I receptors. The mutually inhibitory interaction between MH1 and MH2 domains can be attenuated by receptor-mediated phosphorylation of the SSxS sequence to initiate Smad activation and nucleus accumulation (Massague & Wotton, 2000). Smad4 has an MH1–linker–MH2 structure but lacks the SSxS sequence (Wu et al. 1997), resulting in its not being phosphorylated by receptors. Smad4 primarily uses its MH2 domain to interact with R-Smads. The MH2 domain of Smad6/7 has a similar overall structure to that of Smad4 or R-Smads, but lacks of the SSxS sequence for phosphorylation (Massague et al. 2005). Therefore the inhibitory effect of Smad6/7 is to serve as decoys interfering with Smad–Smad or receptor–Smad interactions.

Nuclear co-factors for the Smad complex

The nuclear co-factors for the activated Smad complex include DNA binding partners and transcriptional co-activators or co-repressors. Different DNA binding partners can determine different choices of target genes. Many DNA binding proteins like Runt-related transcription factor (Runx), O/E1-associated zinc-finger protein (OAZ) and Msh homeobox 1 (Msx1) have been demonstrated to interact with R-Smads in the nucleus (Moustakas & Heldin, 2009). The association with the transcriptional co-activators histone acetyltransferases p300 and CREB-binding protein (CBP) enhances Smad-dependent BMP signalling (Ross & Hill, 2008). On the other hand, the homeodomain protein TGIF recruits histone deacetylases (HDACs) to repress Smad-dependent transcription (Wotton et al. 1999).

Smad-independent signal transducers

In addition to the canonical BMP–Smad signalling, activation of BMP receptors can also trigger Smad-independent signalling primarily through the mitogen-activated protein kinase (MAPK) family members, including extracellular signal-regulated kinases1/2 (ERK1/2), p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinases (JNK) (Guo & Wang, 2009). Moreover, phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), protein kinase C (PKC) and the Rho-GTPases are also involved in the Smad-independent signalling transduction (Hay et al. 2001; Ghosh-Choudhury et al. 2002; Theriault et al. 2007; Lee et al. 2009a). These BMP-dependent non-Smad pathways also play important roles in controlling organ function, which are further elaborated below.

The regulators of BMP signalling

Extracellular regulators of ligands

The extracellular regulators for BMP ligands include BMP antagonists and other non-receptor binding proteins (Umulis et al. 2009). These BMP-binding proteins include BMP antagonists, noggin, twisted gastrulation (TSG), follistatin and soluble CR-containing proteins as well as the DAN family of proteins (Lowery & de Caestecker, 2010; Zakin & De Robertis, 2010). Besides, membrane/matrix-associated proteins such as type IV collagen, fibrillin and BMP binding endothelial regulator (BMPER) interact with specific BMP ligands and modify their activity (Heinke et al. 2008). In most cases, these extracellular binding proteins inhibit BMP signalling through sequestering BMPs from association with receptors or by reducing the movement of BMPs between cells. However, the latest findings suggest these proteins can context-dependently either inhibit or promote BMP signalling (Chang et al. 2007; Jara et al. 2009; Umulis et al. 2009; Sato et al. 2014).

A number of studies have shown that expression changes of extracellular regulators of BMP ligands are involved in several cardiovascular injuries and disorders. BMPER controls the BMP activity to regulate the pro-inflammatory phenotype of endothelium and its expression decreases in response to inflammatory stimulation (Helbing et al. 2011). Matrix Gla protein (MGP) was firstly identified as a calcification inhibitor in cartilage and vasculature. Mice with MGP deficiency exhibit widespread vascular calcification (Luo et al. 1997). However, later studies suggest that, depending on its concentration, MGP can either inhibit or enhance the BMP2 and BMP4 activity (Zebboudj et al. 2002; Yao et al. 2006). Gremlin, a DAN family BMP antagonist, can be significantly upregulated by injury and growth factors such as angiotensin II and TGF-β1 to induce VSMC proliferation and migration (Maciel et al. 2008). High levels of basal gremlin expression in lung and increased expression during hypoxia-induced pulmonary vascular remodelling are linked to the pathogenesis of pulmonary arterial hypertension (PAH) (Cahill et al. 2012). However, in some cases, the effects of increased BMP antagonists on disease development should be interpreted in the context of other BMP signalling components. For example, the increased gremlin expression in the vascular media in rat models of, and patients with, vascular calcification is associated with increased BMP2 expression (Jara et al. 2009). Moreover, in murine and human atherosclerotic vessels, BMP2/4 and gremlin are co-expressed in VSMCs to modulate cross-talk between vascular and immune cells (Sato et al. 2014). In another example, the BMP antagonists follistatin, noggin and matrix Gla protein are found to be co-expressed with BMP4 in ECs exposed to unstable flow (Chang et al. 2007), suggesting an impact of BMP antagonists to minimize the inflammatory response.

Ubiquitin ligases for BMP receptor and Smad degradation

BMP receptors and Smad proteins are regulated by the ubiquitin–proteasome pathway. Smad specific E3 ubiquitin protein ligase 1 (Smurf1), Smad specific E3 ubiquitin protein ligase 2 (Smurf2) and neural precursor cell expressed developmentally down-regulated protein 4-2 (NEDD4-2) from the homologous to the E6-AP carboxyl terminus (HECT) type E3 ligase family can negatively regulate BMP signalling by physically interacting with both R-Smad and Smad4 for degradation (Zhu et al. 1999; Zhang et al. 2001; Moren et al. 2005). Smurfs also interact with I-Smads to induce their nuclear export. In addition, Smurfs promote the association of I-Smads with BMP type I receptors to induce ubiquitination and degradation of these receptors (Murakami et al. 2003). Elevated expression of Smurf1 has been reported in pulmonary arteries of the PAH animal model (Murakami et al. 2010). Smurf1 induction triggers degradation of BMP receptors and promotes vascular cell proliferation and remodelling in PAH. In cultured cardiomyocytes, Smad6/Smurf1 complex formation by BMP2 antagonizes TGF-β1/Rho-associated kinase enhanced cardiac fibrotic signalling (Wang et al. 2012). These studies suggest that targeting ubiquitin ligases for BMP signalling could be a strategy against the development of cardiovascular diseases.

Proteins interact with BMP receptors and Smads

The interacting proteins associated with BMP receptor and Smads may interfere with BMP signalling. The type I protein kinase G (PKGI), an important mediator of vasodilatation, is reported to interact with BMPRII and Smad1/4/5 (Schwappacher et al. 2009). More interestingly, PKGI also phosphorylates BMPRII (Schwappacher et al. 2009). PKGI enhances the BMP signalling via two sequential mechanisms: (1) at the cell membrane, upon BMP2 stimulation, PKGI dissociates from the BMPRII to interact with phosphorylated Smad1/5; the latter is subsequently translocated into the nucleus; and (2) PKGI binds to the Smad complex and TFII-I to promote the transcription of BMP target genes. A subsequent study by the same group (Schwappacher et al. 2013) unravelled the molecular mechanism underpinning the beneficial effects of cyclic GMP-elevating agents in PAH. To maintain pulmonary artery smooth muscle cells (PASMCs) in a differentiated but low proliferative state, PKGI is required for BMP signalling transduction to Smad1/5/8. Other proteins have also been reported to interact with BMP receptors but no clear information is available about their involvement in BMP-associated cardiovascular diseases. TrkC, a tyrosine kinase receptor for neurotrophin-3, is found to directly bind to BMPRII, and to interfere with its interaction with the BMP type I receptor (Jin et al. 2007). Induction of TrkC expression is reported to positively regulate the response of VSMCs to injury (Donovan et al. 1995). It will be of interest to know whether this regulation involves the inhibition of BMP signalling. Ror2, another tyrosine kinase receptor, forms a ligand-independent heteromeric complex with ALK6 to inhibit Smad1/5 signalling (Sammar et al. 2004).

Cross-talk signalling pathways

BMP signalling cross-talks with many other pathways including the MAPK, PI3K/Akt, Wnt/β-catenin and Notch pathways (Guo & Wang, 2009), which modulate the function of BMP signalling. One important basis for the communication between BMP and MAPK/Wnt signalling is the phosphorylation of Smad1 linker region by MAPK and glycogen synthase kinase 3β (GSK3β) (Fuentealba et al. 2007; Sapkota et al. 2007). Phosphorylation of Smad1 linker region creates a docking site for the Smad1/5-specific E3 ubiquitin ligase Smurf1, which inhibits BMP signalling by Smad1/5 degradation. Although there are extensive studies on this signalling cross-talk during development and in cancer and stem cell biology, only a few studies suggest such cross-talk in the cardiovascular system. One example for the cooperation of BMP and Wnt is that the migration of cardiac progenitor cells requires the integration of BMP2 and Wnt3a signals by Smad1 (Song et al. 2014). Fibroblast growth factor (FGF) signalling through MAPK opposes BMP signalling via Smad1 inhibitory phosphorylation (Kretzschmar et al. 1997a). However, FGF is reported to promote BMP signalling in the outflow tract myocardium where it upregulates BMP4 expression to regulate the formation of outflow tract valve primordium (Zhang et al. 2010). Further detailed studies on the regulation of BMP activity by cross-talking to other signalling pathways will enhance our understanding of the dynamics of BMP signalling activity.

Mechanical forces

Mechanical forces are important regulators of the cardiovascular function, including VSMC phenotype, atherogenesis and blood pressure. ECs have been widely studied as a typical example of the impact of mechanical forces (Chien et al. 1998). ECs are constantly exposed to blood flow to experience shear stress, which participates in vascular tone modulation, vascular wall remodelling, and inflammatory reactions by regulating the EC quiescent phenotype (Traub & Berk, 1998; Li et al. 2005). BMP signalling is mechanosensitive. Oscillatory shear stress (OSS) up-regulates endothelial BMP4 protein expression, whereas laminar shear stress (LSS) reduces it (Sorescu et al. 2003). Mechanistically, the RNA-binding protein HuR is involved in the up-regulation of BMP4 expression under OSS, while the activated cyclic AMP/protein kinase A (PKA) in LSS down-regulates BMP4 expression (Csiszar et al. 2007; Rhee et al. 2010). Functionally, through stimulating the release of reactive oxygen species (ROS) derived from the NOX1-dependent NADPH oxidase 1, OSS-induced BMP4 elevates ICAM-1 expression and monocyte adhesion, thus contributing to EC inflammation (Sorescu et al. 2004). Moreover, altered haemodynamics (pulsatile shear stress) can stimulate the expression of the aortic valve leaflet endothelial adhesion molecules VCAM-1 and ICAM-1 via a BMP4-dependent mechanism (Sucosky et al. 2009). While EC-derived overproduction of BMP4 is generally pro-inflammatory, EC-derived BMP4 acting on VSMCs seems to be beneficial in intimal hyperplasia. High shear stress induced by placement in baboons of a distal femoral arteriovenous fistula increases the expression of EC BMP4, which inhibits VSMC proliferation to promote high shear stress-mediated graft neointimal atrophy (Hsieh et al. 2006).

Apart from the mechanical stimulation of BMP4, BMP receptors and Smad proteins have also been implicated in mechano-transduction. Ankeny and colleagues observed that, in calcified human aortic valves, Smad1, Smad5 and Smad8 are highly activated in the calcified fibrosa endothelium together with a low expression of BMP antagonists and inhibitory Smad6 (Ankeny et al. 2011). Zhou and colleagues subsequently demonstrated that OSS can force-specifically activate Smad1/5 to cause cell cycle progression in ECs (Zhou et al. 2012). It is interesting to note that the BMP4-specific antagonist noggin cannot inhibit OSS-induced activation of EC Smad1/5. A later study showed that OSS activates EC Smad1/5 probably through BMP receptor–integrin interaction (Zhou et al. 2013). OSS induces a sustained ALK6-αvβ3 integrin interaction, which signals through the Shc/FAK/ERK pathway, leading to the activation of the Runx2/mTOR/p70S6K cascade and thus promoting EC proliferation.

In addition to shear stress-regulated BMP activity, studies on mechanical stretch implicate pathophysiological roles of BMPs in aortic valve and heart disease. Elevated cyclic stretch-induced aortic valve calcification is BMP-dependent (Balachandran et al. 2010). In response to the magnitude of applied strain in valvular cells but not vascular cells, BMP2/4 and TGF-β1 mRNA expression is significantly altered (Ferdous et al. 2013). A more recent study identifies BMP2 as a new autocrine/paracrine factor that regulates mechano-transduction in cardiomyocytes and adaptation to increased mechanical stretch (Tokola et al. 2015). Similar to the effects of mechanical stretch on cultured cardiac myocytes, BMP2 increases the expression of B-type natriuretic peptide (BNP) and atrial natriuretic peptide (ANP), and activates transcription factor GATA-4.

Angiotensin II type 1 receptor (AT₁R) is a pivotal G-protein coupled receptor in regulating cardiovascular homeostasis. Previous studies revealed that mechanical stress can activate AT₁R in cardiomyocytes without the involvement of angiotensin II (Zou et al. 2004). Recent data demonstrate that the AT₁R subtype A is the mechanosensor in VSMCs from mouse mesenteric and renal arteries that plays a key role in the regulation of vascular contraction (Schleifenbaum et al. 2014). Angiotensin II acting through AT₁R is likely to be involved in stimulating Smad proteins like Smad2 and Smad4 in VSMCs, fibroblasts and cardiomyocytes (Hao et al. 2000; Rosenkranz, 2004; Rodriguez-Vita et al. 2005), thus explaining its profibrotic effects by interplay with TGF-β signalling. Studies from our laboratory and others have found that AT₁R blockers inhibit the expression of BMP4 in ECs and BMP2 in VSMCs (Armstrong et al. 2011; Lau et al. 2013). Moreover, several studies on shear stress show the elevated AT₁R expression in the inner, athero-prone region of the mouse aortic arch (Ramkhelawon et al. 2009). In cultured ECs, OSS up-regulates AT₁R expression, while LSS down-regulates AT₁R expression in PKA- and PKG-dependent mechanisms (Ramkhelawon et al. 2009). In addition, AT₁R-triggered signalling during shear stress activates ERK (Barauna et al. 2013; Ramkhelawon et al. 2013). With regard to the observation that AT₁R signalling utilizes Smad proteins, and the similar expression pattern between AT₁R and BMP–Smad and common upstream regulator (PKA) and downstream kinase (ERK) in shear stress, we propose that AT₁R signalling may interact with the BMP–Smad pathway. Therefore, it is worthwhile to investigate the putative cross-talk between AT₁R and BMP signalling in response to mechanical stimulation.

Pro-inflammatory stimuli

BMP signalling itself is pro-inflammatory in many cell types. But the BMPRII-mediated anti-inflammatory and anti-atherogenic effects are also reported in ECs (Kim et al. 2013). Furthermore, in PAH, BMP2/4 activation is anti-inflammatory in both ECs and VSMCs (Burton et al. 2011; Wang et al. 2012). How do pro-inflammatory stimuli activate BMPs to render them a downstream effector? In coronary arterial ECs (CAECs), tumour necrosis factor-α stimulation increases BMP2 mRNA and protein expression through the H₂O₂-dependent NF-κΒ activation (Csiszar et al. 2005). Also in CAECs, Toll-like receptor 2 and 4 mediate ox-LDL-induced BMP2 expression, suggesting that the effect of these two immunoreceptors in the pathological process of atherosclerotic vascular calcification may involve BMP2 activation (Su et al. 2011). In VSMCs of saphenous veins, high glucose stimulates expression of the RAGE (receptor for advanced glycation end-products) ligand high mobility group box 1 (HMGB1), which contributes to vascular calcification through NF-κB-dependent up-regulation of BMP2 (Wang et al. 2013). Saturated fatty acids can activate macrophages and induce vascular inflammation. A recent study has linked saturated fatty acids to the progression of vascular diseases by demonstrating that palmitate stimulates macrophage secretion of BMP2/4, which acts on VSMCs to increase migration (Chung et al. 2012). In summary, pro-inflammatory signals primarily transduce to nuclear factor NF-κB, the master regulator of inflammation, to increase BMP ligand expression. IL-6 also regulates BMP signalling. In PASMCs, IL-6 and mutational BMPRII signalling form a negative feedback loop to result in a poor regulation of cytokines by BMPRII mutation (Hagen et al. 2007). IL-6 was also reported to down-regulate BMPRII expression through Stat3-microRNA signalling in pulmonary arterial endothelial cells (PAECs) (Brock et al. 2009).

MicroRNAs

MicroRNAs (miRNAs) are small non-coding RNAs that importantly participate in post-transcriptional regulation of gene expression. MiRNAs are critically involved in the pathogenesis of cardiovascular disease, and targeting important miRNAs may have preventative and reparative therapeutic potential for the treatment of cardiovascular diseases (Quiat & Olson, 2013). A number of miRNAs have been demonstrated to impact on cardiovascular function through targeting the BMP pathway. The miR-26a was firstly identified as a new regulator of VSMC differentiation, proliferation, apoptosis and migration through affecting the expression of Smad1 and Smad4 proteins (Leeper et al. 2011). A subsequent study showed that miR-26a regulates both pathological and physiological angiogenesis via the BMP–Smad1-dependent mechanism in ECs (Icli et al. 2013). In PAH, down-regulation of BMPRII protein but not mRNA levels suggests that a post-transcriptional regulatory mechanism of the BMPRII gene may be involved. A computational algorithm on the BMPRII gene together with experimental data show that BMPRII mRNA 3′-untranslated region can be targeted by miR-17-5 and miR-20a from the miRNA 17/92 cluster, which works downstream of IL-6–Stat3 signalling (Brock et al. 2009). Also in PAH, miR-21 works as a modifying miRNA that integrates down-regulated BMPRII and increased Rho/Rho-kinase pathological signalling to control PAH. (Parikh et al. 2012). BMP2 is well known for its major regulatory role in vascular calcification in VSMCs; a recent study revealed that BMP2-dependent aortic valvular calcification is regulated by miRNA-141 and also demonstrated marked attenuation of miR-141 expression in patients with bicuspid aortic valve-associated aortic stenosis (Yanagawa et al. 2012). With the advancement of bioinformatics and molecular biology technologies, more miRNAs will be identified as novel regulators of cardiovascular function through targeting BMP signalling. In addition, the reported cardiovascular regulatory miRNAs that can target BMP signalling in other tissues and organs may attract further exploration of whether these miRNAs exert cardiovascular effects through similar molecular mechanisms involving the BMP signalling.

Small-molecule drugs

Small-molecule drugs constitute the vast majority and diversity of drugs in the global marketplace. Because of the advantages small-molecule drugs offer, the discovery and development of small-molecule drugs targeting BMP signalling will benefit patients with diseases involving abnormal BMP signalling and assist basic researchers interested in the elucidation of molecular regulation of this pathway. FK506, an immunosuppressive drug, is mainly prescribed to patients after allogeneic organ transplant. Through a transcriptional high-throughput luciferase reporter assay to screen 3756 FDA-approved drugs and bioactive compounds for induction of BMPRII signalling, FK506 was identified as the best responder (Spiekerkoetter et al. 2013). Further studies showed that FK506 corrects dysfunctional BMPRII signalling, rescues endothelial dysfunction, and thus reverses PAH (Spiekerkoetter et al. 2013). Statins, the widely used cholesterol-lowering drugs, were reported to modulate the BMP pathway by upregulating BMPER and reducing BMP4 expression (Helbing et al. 2010). BMP4 down-regulation and BMPER up-regulation contribute to the anti-inflammatory pleiotropic effects of statins. Boldine, a potent natural antioxidant, inhibits the oxidative stress cascade initiated by angiotensin II-stimulated BMP4 to protect endothelial function in type 2 diabetic db/db mice (Lau et al. 2013). As discussed previously, the ubiquitin ligase Smurf1 negatively regulates BMP–Smad signalling. Cao and colleagues used structure-based virtual screening to discover small molecular compounds that disrupt Smurf1–Smad1/5 interaction. The screened selective compounds were shown to facilitate BMP2 responsiveness through decreased Smurf1-mediated Smad1/5 degradation (Cao et al. 2014), thus highlighting the role of Smurf1 as an eligible pharmacological target for increasing BMP signalling. Small-molecule inhibitors for BMP receptors have been screened and functionally assessed in pre-clinical studies. Dorsomorphin (compound C), an AMP-activated protein kinase (AMPK) inhibitor, has been screened as a selective inhibitor for BMP type I receptors (including ALK1, ALK2, ALK3 and ALK6) (Yu et al. 2008b; Wrighton et al. 2009). Besides, dorsomorphin derivatives with higher activity and specificity for BMP type I receptors have also been developed (Cuny et al. 2008; Hao et al. 2010). Despite these compounds having been demonstrated to be potentially effective against some clinical disorders such as iron-hepcidin homeostasis and ossification (Yu et al. 2008a; Yu et al. 2008b), their effects in the regulation of cardiovascular function are currently not well characterized. These inhibitors might have therapeutic potential in cardiac regeneration through directed differentiation of stem cells to cardiomyocytes. The studies on mouse embryonic stem cells show that inhibition of BMP type I receptors with dorsomorphin and its derivative DMH1 increases cardiomyocyte progenitors and promotes cardiac differentiation (Hao et al. 2008; Ao et al. 2012). However, simultaneous inhibition of BMP and AMPK signalling by dorsomorphin makes it more difficult to reveal the proportional contribution of these two pathways to its cardiovascular benefit. Hence, the development of higher-specificity derivatives antagonizing the BMP type I receptor may help advance the BMP-treated pharmacological therapies of cardiovascular diseases.

Direct BMP target genes and regulated effectors

BMP signalling effectors execute the upstream signalling command to functionally alter cellular and organ function. Hence, understanding the regulation of downstream effectors and functional consequences mediated through these effectors will provide a better control of BMP signalling (Fig.2).

Figure 2.

Schematic diagram of BMP regulated effectors involved in the pathogenesis of cardiovascular disorders

Ang-1, angiopoietin 1; VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1; HEY1/HEY2, hairy/enhancer-of-split related with YRPW motif protein 1/2; HES1, hairy and enhancer of split-1; UNC5B, unc-5 homologue B (vascular netrin receptor); GM-SCF, granulocyte–macrophage colony-stimulating factor; miR-145, microRNA-145; TRPC, canonical transient receptor potential channel; ET-1, endothelin 1; ID1, inhibitor of DNA-binding 1; ID3, inhibitor of DNA-binding 3; ApoE, apolipoprotein E; p-eNOS, phosphorylated endothelial nitric oxide synthase; Kv1.5, voltage-gated potassium channel, shaker-related subfamily, member 5; Kv4.3, voltage-gated potassium channel subfamily D member 3; ANP, atrial natriuretic peptide; BNP, B-type/brain natriuretic peptide; BMP4, bone morphogenetic protein 4; NOX4, NADPH oxidase 4; NOX1, NADPH oxidase 1; ROS, reactive oxygen species; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular adhesion molecule 1; SM22, transgelin; miR-21, microRNA-21; p21, cyclin-dependent kinase inhibitor 1; Runx2, runt-related transcription factor 2; Pit-1, sodium-dependent phosphate transporter 1.

BMP–Smad target genes

Numerous BMP–Smad target genes have been identified. Here we discuss those BMP target genes that are importantly involved in the regulation of cardiovascular function. BMP–Smad signalling is involved in angiogenesis through regulating several key genes. In particular, ALK1 as an EC specific type I receptor is functionally responsible for human hereditary haemorrhagic telangiectasia (HHT) type II (Dupuis-Girod et al. 2010). Microarray analysis in human umbilical vein endothelial cells shows that ALK1 stimulation can up-regulate Smad6, Smad7, ID1, ID2, endoglin, Stat1 and interleukin 1 receptor-like 1 while down-regulating Smad1, CXCR4, Ephrin-A1 and plakoglobin (Ota et al. 2002). BMP9/ALK1 signalling cooperates with the Notch pathway to inhibit angiogenesis through Smad1/5/8-induced expression of Notch target genes HES1, HEY1 and HEY2 (Larrivee et al. 2012). ALK1 also reduces VEGF responses and tip cell specification by increasing the expression of UNC5B and VEGFR1 (Larrivee et al. 2012). The BMP antagonist Drm, a member the Dan family of BMP antagonists, exerts pro-angiogenic activity through angiopoietin-1, a vascular growth factor playing a role in embryonic and postnatal angiogenesis (Mitola et al. 2008). In microvascular ECs BMP4/7/9 signals through the BMPRII–Smad pathway to down-regulate apelin expression, thus impairing hypoxia-induced EC proliferation (Poirier et al. 2012). However, in contrast to the inhibitory role of BMPs in hypoxia-induced proliferation, BMP4 was reported to promote proliferation and migration of ECs via stimulation of VEGFA/VEGFR2 and angiopoietin-1/Tie2 signalling (Suzuki et al. 2008). These findings suggest that BMP signalling may context-dependently activate endothelium or turn ECs into quiescent states.

Many BMP signalling targets have been reported in PAH. Endothelin-1, a potent vasoconstrictor and one of the mediators of PAH, was found to be increased by BMP9 in pulmonary microvascular ECs (Star et al. 2010). This study provides a potential mechanism underlying endothelin-1 elevation in PAH. ID1 protein is a well-defined target gene of BMPs in ECs (Valdimarsdottir et al. 2002). On the other hand, BMP2 and BMP4 stimulate the phosphorylation and activity of endothelial nitric oxide synthase (eNOS) through the BMPRII–PKA pathway to mediate the effects of increased proliferation, survival and migration in PAECs (Gangopahyay et al. 2011). Recruitment of inflammatory cells to the pulmonary vascular wall contributes to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) (Tuder et al. 1994). Induction of endothelial GM-CSF translation and recruitment of macrophages resulting from reduced BMPR2 expression were linked to exacerbating IPAH (Sawada et al. 2014). In PASMCs, ID1 and ID3 are the critical downstream effectors of BMP signalling and they inhibit cell cycle progression to regulate the proliferation of PASMCs (Yang et al. 2013). But this study does not show whether forced ID1 and ID3 expression is sufficient to rescue BMPRII mutation-caused PASMC proliferation. In addition, BMP2/7 inhibits proliferation of VSMCs to maintain a quiescent phenotype by the cyclin-dependent kinase-2 inhibitor, p21 (Dorai & Sampath, 2001; Wong et al. 2003). Ion channels are the key components in rapidly changing biological processes in cardiovascular cells. Both BMP axis and voltage-gated K⁺ channels (K_v) are down-regulated in PAH (McMurtry et al. 2004). Expression of PASMC K_v channels is found to be controlled by BMP2–BMPRII signalling in PAH (Fantozzi et al. 2006; Young et al. 2006). Elevated intracellular Ca²⁺ concentration is a key signal for PASMC proliferation and contraction. BMP2-induced inhibition of canonical transient receptor potential channel (TRPC) expression decreases Ca²⁺ signalling in rat PASMCs (Zhang et al. 2013), which may partly explain the beneficial role of BMP signalling in PAH.

Smad-independent effectors

ROS and MAPK signalling are two critical mediators for the Smad-independent signalling in cardiovascular cells, especially ECs (Fig.3). BMP2 and BMP4 can directly induce ROS overproduction, which subsequently impairs endothelium-dependent vasodilatation (Csiszar et al. 2006; Miriyala et al. 2006; Wong et al. 2010). Elevated BMP4 expression in relation to increased oxidative stress was demonstrated in arteries from spontaneously hypertensive rats and hypertensive patients and is closely associated with impaired endothelial function, and BMP4-stimulated oxidative stress involves p38–cyclooxygenase-2-dependent mechanisms (Wong et al. 2010). BMP4 triggers ROS over-generation in primary cultured mouse ECs leading to EC apoptosis through activation of the p38/JNK pathway (Tian et al. 2012). Inhibition of BMP4 with noggin and its receptor ALK3 by viral silencing restores arterial endothelial function via suppression of vascular ROS in type 2 diabetic mice (Zhang et al. 2014). Besides, BMP4 was also shown to contribute to venous endothelial dysfunction diabetic patients undergoing coronary revascularization (Hu et al. 2013). Protein carbonylation plays a critical role in mediating oxidant signalling in various cells, but whether it mediates endothelial dysfunction is unclear. Recent work done by us suggests that BMP4- or diabetes-mediated endothelial dysfunction is partly triggered through protein carbonylation and blockade of this metal-catalysed protein oxidation can be considered as an alternative therapeutic strategy to alleviate diabetic vasculopathy (Wong et al. 2014). In VSMCs, BMP2 also increases oxidative stress and endoplasmic reticulum stress through NADPH oxidase activation and overexpression of Runx2 and sodium-dependent phosphate co-transporter Pit-1, and consequently promotes vascular calcification (Li et al. 2008; Liberman et al. 2011). In both human and murine PASMCs, independent of Smad1/5/8, BMP2 rapidly signals to PPARγ to upregulate expression of apoE, which mediates an antiproliferative effect to reverse PAH (Hansmann et al. 2008). In cardiomyocytes, BMP4 promotes cellular apoptosis to mediate myocardial ischaemic injury through a JNK-dependent pathway (Pachori et al. 2010). In experimentally induced pathological cardiac hypertrophy, BMP4 increases NADPH oxidase 4 (NOX4) expression and associated ROS cause cardiomyocyte hypertrophy and apoptosis (Sun et al. 2013a). More recently, studies from the same group also identified more BMP-signalling downstream targets including down-regulated K_v4.3 potassium channels and upregulated BMP4 itself, which contribute to BMP4-induced pathological cardiac hypertrophy (Sun et al. 2013b; Wang et al. 2015). In conclusion, strategies against oxidative stress and MAPK signalling will benefit cardiovascular function under conditions of exaggerated BMP signalling such as hypertension and diabetes.

Figure 3.

Proposed non-Smad cellular mechanisms for BMP4-triggered endothelial cell dysfunction and apoptosis

BMP4 production in endothelial cells can be induced or exaggerated by several risk factors such as oscillatory shear stress, angiotensin II, oxidized LDL and high glucose. BMP4 stimulates the BMP receptor IA to increase expression and activity of oxidative stress-dependent COX-2 via a p38 MAPK-dependent mechanism. The resultant elevated BMP4-stimulated NADPH oxidase-derived superoxide anions rapidly inactivate NO to reduce endothelium-dependent relaxations, and it can also stimulate the activity of the up-regulated COX-2 to release greater amounts of constrictive prostaglandins to evoke endothelium-dependent contraction through interaction with TP receptors in VSMCs. In addition, BMP4-elevated superoxide anions can sequentially activate p38 MAPK and JNK, upstream of caspase 3 activation, to induce endothelial cell apoptosis. Noggin is the BMP4 antagonist. BMP4, bone morphogenetic protein 4; COX-2, cyclooxygenase-2; NO, nitric oxide; TP, thromboxane prostanoid; VSMCs, vascular smooth muscle cells.

BMP-regulated miRNAs

Apart from serving as critical regulators of BMP signalling, miRNAs are also crucial effectors downstream of BMP signalling. Several miRNAs had been reported to mediate the effect of BMPs on cardiovascular cells. A seminal study showed that miR-21 mediates the TGF-β/BMPs-induced contractile phenotype in VSMCs through down-regulating the negative regulator of smooth muscle contractile genes, PDCD4 (programmed cell death 4) (Davis et al. 2008). A later study uncovered a new regulatory mechanism of the VSMC phenotype by the BMP4–miR-21 axis through DOCK (dedicator of cytokinesis) family proteins (Kang et al. 2012). In cardiac progenitor cells, BMP signalling regulates myocardial differentiation via regulation of the miRNA-17-92 cluster (Wang et al. 2010). As described above, the miRNA-17/92 cluster targets BMPRII through miR-17-5p and miR-20a. It is of interest to note these two individual miRNAs are also found to be the direct effectors that repress the expression of cardiac progenitor genes Isl1 and Tbx1 resulting in enhanced myocardial differentiation. Mutations of BMPRII have been identified in approximately 70% of patients with the heritable form of PAH (hPAH). The PAH smooth muscle cells express a greater level of miR-145 as a result of mutational BMPRII signalling (Caruso et al. 2012). All these studies further support the notion that inhibitors or mimics of important miRNAs can become promising therapeutic targets against cardiovascular dysfunction.

Conclusion and future perspectives

BMPs exert either harmful or beneficial cardiovascular effects depending on the types of ligands, interactive receptors and associated intracellular transduction signalling pathways (Smads and non-Smads) involved. For example, BMP4 up-regulation in ECs stimulated by disturbed blood flow is among the critical factors mediating vascular inflammation and atherosclerotic progression as well as contributing to endothelial dysfunction in hypertension and diabetes. In addition, the tissue content of BMPs is modulated by their endogenous antagonists; BMP receptors are regulated or inhibited by miRNAs or small-molecule drugs in ECs; and Smad signalling is targeted by ubiquitin ligases, miRNAs or laminar shear stress (Fig.1). In BMP-initiated non-Smad pathways, targeting both upstream regulators such as proinflammatory stimuli (e.g. angiotensin II, high glucose) or disturbed shear stress and downstream effectors (cyclooxygenase-2, MAPK and apoptotic-mediating enzymes) of the BMP signalling (Fig.3) might be equally effective to reverse vascular dysfunction in cardio-metabolic pathogenesis through screening small-molecule drugs with therapeutic potential for the treatment of cardio-metabolic diseases. Finally, deeper understanding of the regulatory mechanisms of production and secretion of endogenous antagonists of BMPs in conjuncture with cardiovascular pathogenesis will provide additional opportunities for BMP-based therapies.

Glossary

ALK

activing receptor-like kinase

BMP

bone morphogenetic protein

EC

endothelial cell

ERK

extracellular signal-regulated kinase

GDF

growth and differentiation factor

JNK

c-Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

PAEC

pulmonary arterial endothelial cell

PAH

pulmonary arterial hypertension

PASMC

pulmonary artery smooth muscle cell

ROS

reactive oxygen species

Smurf

Smad-specific E3 ubiquitin protein ligase

TGF-β

transforming growth factor-β

VSMC

vascular smooth muscle cell

Biographies

Jiang-Yun Luo is a final year PhD candidate at the Chinese University of Hong Kong interested in the transcriptional regulation of endothelial gene expression.

Yang Zhang is a post-doctoral research fellow at Harvard Medical School studying the role of BMP4 in diabetic endothelial dysfunction.

LiWang is a post-doctoral research fellowat the Chinese University of Hong Kong studying the microRNA regulation of BMP–Smad expression and activity.

YuHuang is the professor of biomedical sciences at the Chinese University of Hong Kong and his research focus is mainly on elucidation of molecular mechanisms involved in vascular function/dysfunction in cardio-metabolic diseases (http://www.cuhk.edu.hk/proj/HuangLab/).

Additional information

Competing interests

There are no competing financial interests.

Funding

We would like to acknowledge the support from the Hong Kong Research Grants Council (CUHK2/CRF/12 G and T12-705/11) and the Natural Science Foundation of China (91339117 and 2012CB517805).