BBSome: A New Player in Hypertension and Other Cardiovascular Risks

Abstract

The BBSome is an octameric protein complex involved in Bardet Biedl syndrome (BBS), a human pleiotropic, autosomal recessive condition. BBS patients display various clinical features including obesity, hypertension and renal abnormalities. Association studies have also linked the BBS genes to hypertension and other cardiovascular risks in the general population. The BBSome was originally associated with the function of cilia, a highly specialized organelle that extend from the cell membrane of most vertebrate cells. However, subsequent studies have implicated the BBSome in the control of a myriad of other cellular processes not related to cilia including cell membrane localization of receptors and gene expression. The development of animal models of BBS such as mouse lines lacking various components of the BBSome and associated proteins has facilitated studying their role in the control of cardiovascular function and deciphering the pathophysiological mechanisms responsible for the cardiovascular aberrations associated with BBS. These studies revealed the importance of the neuronal, renal, vascular, and cardiac BBSome in the regulation of blood pressure, renal function, vascular reactivity, and cardiac development. The BBSome has also emerged as a critical regulator of key systems involved in cardiovascular control including the renin-angiotensin system. Better understanding of the influence of the BBSome on the molecular and physiological processes relevant to cardiovascular health and disease has the potential of identifying novel mechanisms underlying hypertension and other cardiovascular risks.

Introduction

Studies of monogenic disorders have been instrumental in uncovering novel genes and deciphering the molecular mechanisms underlying common diseases including hypertension.¹ Bardet-Biedl Syndrome (BBS) is a pleiotropic autosomal recessive disorder characterized by the presence of several clinical features including cardiovascular risks such as obesity, renal abnormalities and hypertension.² Studies aimed at understanding the pathophysiological processes involved in BBS has led to the identification of 24 causative genes (BBS1–BBS24), with mutations in any of these genes resulting in the same general phenotypes.^{3, 4} Mutations in BBS1 and BBS10 genes are the most prevalent, accounting for about 70% of cases in populations of northern European descent. Importantly, variants of several BBS genes were found to increase susceptibility to obesity and hypertension in non-BBS individuals, implicating these genes in the development of common forms of cardiometabolic diseases.^5–7 BBS mouse models generated by introducing mutations (e.g., Bbs1^M390R) or knocking out of various Bbs genes exhibit major components of the human phenotype, including obesity, hypertension and other cardiovascular risks.^8–10 Consistent with this, Bbs genes were found to be expressed in various tissues and cells that are critical for the control of blood pressure and other cardiovascular parameters.

The overlapping phenotypes arising from mutations in such a large number of BBS genes remained puzzling until the discovery that various BBS proteins interact together to form a complex termed the BBSome, which consist of eight conserved BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBS18)¹¹ (Figure 1). This protein complex has emerged as an important player in the control of blood pressure and development of hypertension as well as renal, vascular and cardiac alterations. In this review, we discuss the role of the BBSome in cardiovascular regulation as well as the development of hypertension and other cardiovascular risks. To understand the mechanisms underlying BBSome regulation of blood pressure and other cardiovascular functions in health and disease we will first discuss the composition and formation of the BBSome and the various cellular and molecular processes controlled by this complex.

Figure 1:

Schematic representation of the BBSome and its cellular effects. The assembly of the 8 units of the BBSome is mediated by the BBS chaperonins complex (BBScc) that contain BBS6, BBS10 and BBS12. A major role of the BBSome relate to the trafficking of cargos to and from cilia including G protein-coupled receptors through a process that involve BBS3. The BBSome has also emerged as a critical mechanism for the cell membrane localization of various receptors including the leptin receptor, insulin receptor and G protein-coupled receptors in a manner independent of its ciliary function. In addition, the BBSome has been implicated in the regulation of several other cellular processes including cytoskeleton dynamic, RhoA activity, proteasomal activity and gene expression. The dashed line indicates a potential, but not proven, function of the BBSome.

BBSome FORMATION

The BBSome is assembled in a regulated stepwise process, with the first step involving the formation of BBS2 and BBS7¹². The addition of BBS9 lead to the formation of the core complex. This is followed by the addition of BBS5 and BBS8. Of note, functional subcomplexes are formed when genes that encode BBSome proteins are mutated.^{13, 14} For instance, absence of BBS8 results in the formation of a BBS subcomplex consisting of BBS2, BBS5, BBS7, and BBS9.¹⁵ Three other BBS proteins (BBS6, BBS10, BBS12) were found to form another complex with the CCT (Chaperonin-containing tailless complex polypeptide 1, also known as TRiC, tailless complex polypeptide 1 ring complex) family of group II chaperonins (Figure 1).¹⁶ This chaperonin BBS complex mediates BBSome assembly in an ATP-dependent manner. The role and function of other BBS proteins not associated with the BBSome (BBS11, BBS13, BBS15-16, BBS19-24) remain to be determined. The possibility that BBS proteins including components of the BBSome and BBS chaperonins complex may possess subunit-specific functions cannot be excluded.

CILIARY FUNCTION OF THE BBSome

One of the best characterized function of the BBSome relates to its involvement in the control of ciliary function. A cilium, which is present on virtually all cell types in the human body, is an evolutionarily conserved, hair-like structure emanating from the cell surface and consisting of a microtubule axoneme surrounded by specialized ciliary membrane (Figure 1). The spectrum of diseases (called ciliopathies) that arises from defects in the cilium highlights the importance of this organelle for human health, including cardiovascular well-being. Cilia are equipped with various receptors and signaling molecules allowing them to sense extracellular cues, and the transduction of those cues into cellular signaling ultimately affects a wide range of cellular processes including gene transcription, cell division, and differentiation.¹⁷

The various components of the cilium are put together and maintained by a multiprotein machinery termed intraflagellar transport (IFT) that moves cargo vesicles, such as structural components, receptors, and signaling molecules, into (anterograde) and from (retrograde) the cilium.^17–19 The anterograde transport of particles to the ciliary tip is regulated by the IFT complex B, in association with kinesin motors. On the other hand, the retrograde transport of particles from the tip of the cilium is mediated by the IFT complex A. Because cilia are devoid of protein synthesis, IFT machinery is required for the formation, maintenance, and function of cilia.^19–21

The BBSome play an important role in coordinating movement rates of different particles of the IFT. Indeed, the BBSome act as a membrane coat complex that enlists resident proteins to cilia in concert with BBS3, a small Arf-family GTPase that controls BBSome recruitment to the membrane and BBSome entry to cilia.¹¹ In addition, BBS3 was recently found to promote the exit of specific cargo from cilia via the BBSome²². BBS17, also known as Leucine zipper transcription factor-like 1, controls the trafficking and dynamics of the BBSome in cilia and promotes BBS3 to target to the basal body, which determines the amount of the BBSome available for integration into anterograde IFT trains for entering cilia.^{23, 24} It should be noted, however, that although absence of the BBSome causes subtle ciliary changes, cilia are formed and generally appear normally shaped indicating that the BBSome and BBS proteins are not required for ciliogenesis.^{11, 25–27} On the other hand, BBSome deficiency disrupt the trafficking to cilia of certain proteins such as polycystic-1 (PC1)²⁸ and various G protein-coupled receptors including somatostatin receptor 3, melanocortin concentrating hormone receptor 1 and neuropeptide Y2 receptor²⁹. This highlights the importance of the BBSome for the ciliary localization of different proteins and receptors.

In the kidney, primary cilia are present on most cells of the nephron and on the apical surface of the epithelium extending into the tubule lumen. Ciliary defects have been implicated in epithelial repair and the pathogenesis of different forms of cystic kidney diseases that are commonly associated with the development of hypertension. Modeling ciliopathy in rats through deletion of the Alsm1 (Alström syndrome 1) gene leads to hypertension.³⁰ However, this phenotype appears to be independent of cilia, but seems to involve defects in Na⁺K⁺/Cl⁻ cotransporter (NKCC2) endocytosis, leading to an increase in NaCl reabsorption. Interestingly, male mice bearing inducible nephron-specific ablation of cilia through genetic deletion of the ift88 gene were found to develop a relative hypotension prior to cyst formation.³¹ These mice also displayed increased nitric oxide production and exaggerated salt-induced natriuresis and hypertension. In the vasculature, cilia present in the luminal side of endothelial cells serve as sensors of the blood flow, whereas cilia of smooth muscle cells may act as strain sensors.³² Abnormal vascular cilia function has been suggested to contribute to the development of vascular defects including hypertension.^{32, 33} In the heart, different types of cilia are expressed in a spatiotemporal manner to control various aspects of cardiogenesis, including the left-right patterning and correct looping of the heart. As a consequence, ciliopathies are commonly associated with congenital heart disease and situs inversus, in which cardiac looping is randomized.³⁴

NON-CIALIARY ROLES OF THE BBSome

Studies demonstrating the expression of BBS genes in non-ciliated cells represent the first indication that the BBS proteins may be involved in the control of other functions beyond the cilium.^{35, 36} This idea was further supported by the observation that BBS proteins have extra-ciliary localization^37–39 and the finding that zebrafish lacking various Bbs genes display significant delay in retrograde intracellular transport of melanosomes, a process that does not involve cilia.⁴⁰ Studies in recent years have implicated the BBSome in the control of several cellular processes (Figure 1).

Leptin Receptor Trafficking

Investigation of the mechanisms underlying the obesity phenotype and metabolic defects associated with BBS led to the identification of an important role for the BBSome in the cell surface localization of various receptors. For instance, a component of the BBSome (BBS1) was found to interact physically with the long signaling form of the leptin receptor (LepRb) and mediates its trafficking to the plasma membrane.³⁸ No other components of the BBSome interacted with the LepRb. Interestingly, the unique C-terminal cytoplasmic domain of LepRb was sufficient to interact with BBS1. Moreover, the BBS1 M390R mutation, which is the most common mutation found in human BBS patients and is sufficient to induce BBS phenotypes in a knock-in mouse model⁹, greatly reduced the ability of BBS1 to interact with the LepRb.³⁸ On the other hand, BBS10 was found to promote the stability of the LepRb by enhancing its translation and/or reducing its degradation.⁴¹ Notably, disruption of the BBSome substantially reduced the surface expression of the LepRb which leads to leptin resistance and alteration in the neurocircuit regulating food intake and energy expenditure.^{38, 41, 42}

The role of the BBSome in the trafficking of the LepRb is independent of cilia, as ablation of cilia (through deletion of the Ift88 gene) did not recapitulate the leptin resistance evoked by disruption of the BBSome. Of note, BBS17 was also implicated in leptin sensitivity by regulating LepRb activation of the transcription factor Stat3 (signal transducer and activator of transcription 3).⁴³ Serotonin 5-HT_2C receptor present in hypothalamic neurons is another important regulator of energy homeostasis that depends on the BBSome for its plasma membrane localization.³⁹ Together, these findings demonstrate that defects in the trafficking and signaling of key receptors regulating energy balance may explain the development of obesity in BBS.

Trafficking of other Receptors

The requirement of the BBSome for the handling of the insulin receptor appear to account for the development of insulin resistance and diabetes in BBS. This is supported by the direct interaction between the insulin receptor and various BBS proteins^{41, 44}. Moreover, BBSome deficiency results in significant reduction in the surface expression of the insulin receptor, which translates into reduced signaling capacity. This leads to defects in glucose metabolism and insulin resistance even when the BBS mice are rendered lean through calorie restriction.⁴⁴ The BBSome was also implicated in the cell surface expression of receptors implicated in functions other than metabolism. For instance, the Notch receptor—which is involved in the control of cell differentiation, proliferation, and apoptosis—failed to localize to the plasma membrane in the absence of the BBSome components BBS1 and BBS4.⁴⁵ Interestingly, loss of BBS3 also reduced the plasma membrane localization of the Notch receptor although not to the same extend as BBSome deficiency.

Consistent with their essential role in the transport of receptors to the plasma membrane, BBSome proteins were associated with the secretory pathway. Double immunofluorescent staining and co-immunoprecipitation assays revealed that BBS proteins are present in the late endosome as indicated by their colocalization with markers of this organelle (e.g., Rab7-GTP and CD63).³⁹ Moreover, disruption of the BBSome was found to result in the accumulation of the 5-HT_2C receptor and Notch receptor in the late endosome.^{39, 45} More work is needed to understand the exact mechanisms underlying the sorting of specific cargos to the plasma membrane by the BBSome.

Proteasomal Activity

Proteasome-mediated proteolysis is crucial for maintaining proper cellular levels of many proteins and regulates several signaling pathways including canonical Wnt signaling. Decreasing BBS1 and BBS4 levels was shown to alter proteasomal subunit composition and inhibit its activity.⁴⁶ Moreover, deletion of BBS4 was found to cause defective proteasome-mediated protein clearance leading to the accumulation of β-catenin⁴⁷, a protein involved in the regulation and coordination of cell-cell adhesion and gene expression. BBS11 was reported to function as an E3-ubiquitin ligase.^{48, 49} Zhang et al. showed that both endogenous and exogenous BBS2, when not in the BBSome complex, were ubiquitinated despite inhibition of proteasomal activity.¹² This degradation of free BBS2 seem to involve BBS11. However, the proteasomal function is also regulated by cilia.^{50, 51} Therefore, the direct effect of the BBSome on proteasomal activity remains unclear.

Gene Expression

Identification of nuclear export signals in several BBS proteins predicted that these proteins may play a role in transcriptional regulation. This was confirmed by the demonstration that BBS7 dynamically enters the nucleus and interacts and regulates levels of the nuclear protein RNF2 (ring finger protein 2), a protein-coding gene.⁵² These observations are in agreement with the early finding that two mutations (H323R and T211I) in the BBS7 gene increased the nuclear localization of the BBS7 protein.⁵³ In addition to BBS7, various other BBS proteins were shown to be readily present in the nuclear compartment and that absence of BBS4 significantly increased the level of RNF2. On the other hand, BBS6 was found to interact and modulate the subcellular localization of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling protein Smarcc1a (matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1a), negatively regulating its import into the nucleus.⁵⁴

A role for BBS proteins in transcriptional regulation is further supported by the changes in gene expression described in various tissues of BBS-deficient mice. In the hypothalamus, a significant decrease in proopiomelanocortin gene expression was reported in different mouse models of BBS.^{8, 38, 41} In addition, disruption of the BBSome by Bbs1 gene deletion in smooth muscle cells increased the expression of the vascular angiotensinogen gene, leading to activation of the renin-angiotensin system.⁵⁵ Future studies are warranted to determine whether these changes in gene expression are a primary effect of BBS protein deficiency or a secondary consequence of other changes such as receptor mistrafficking. It is also unclear whether the involvement of BBS proteins in transcription regulation is separate from their role as part of the BBSome.

Other Cellular Functions

Accumulating evidence point to a role for BBS proteins in the regulation of a number of other cellular processes through mechanisms that do not involve cilia. For instance, the BBSome modulate the intracellular machinery implicated in cell migration and tissue repair, as BBSome deficiency leads to upregulation of RhoA expression and activity and reduction in the ubiquitin ligase Culin-3, resulting in impairment in cell migration and wound healing.⁵⁶ Additionally, the BBSome has been reported to be involved in the cytoskeleton organization, cell division, and endoplasmic reticulum stress response.^57–60 However, the regulation of some of these functions such as the actin cytoskeleton dynamic by the BBSome may be related to the role of this complex in cilia function because ciliogenesis depends on the actin filament bundles and networks.

BBSome AND HYPERTENSION

Human Studies

A study involving 152 BBS subjects provided robust evidence that hypertension is more prevalent in individuals with BBS relative to controls matched for sex, age, and body mass index.⁶¹ This is further supported by the demonstration that BBS patients are about 8 times more likely to develop hypertension relative to family members without a BBS gene mutation.⁶² This high prevalence of hypertension and its early onset has led to the idea that hypertension should be considered as a primary phenotype in the diagnosis of BBS.

Consistent with the variable phenotypic expressivity associated with BBS, some variability in the presence and severity of hypertension has been noted. For instance, BBS subjects carrying mutations in the BBS10 gene tended to develop a more pronounced increase in systolic blood pressure.⁶¹ On the other hand, in a 22-year prospective study of a cohort of 46 BBS patients, all BBS genotypes were found to be accompanied by hypertension except for BBS2.⁶³ This is particularly interesting because variants of BBS4 and BBS6, but not BBS2, genes were found to increase the risk of hypertension in a large cohort of Caucasian subjects.⁵ These findings highlight the relevance of BBS genes to common forms of human hypertension. This is further supported by the high prevalence of hypertension in BBS heterozygous carriers, which is estimated to be present in 1% of the general population.⁶⁴

Animals Studies

The development of animal models of BBS has greatly facilitated analysis of the role of the BBSome in the control of blood pressure and the mechanisms underlying BBS-associated hypertension. We demonstrated that various BBS mouse models including Bbs3-, Bbs4-, and Bbs6-null mice have elevated blood pressure, whereas Bbs2 knockout mice do not.^{8, 10} Moreover, mice lacking the Bbs3, Bbs4, and Bbs6 genes, but not the Bbs2 gene, displayed a significant increase in renal sympathetic nerve activity and an exaggerated depressor response to ganglionic blockade, highlighting the role of the sympathetic nervous system in BBS-associated hypertension. The BBS2 mouse model provides a unique opportunity to gain mechanistic insights into the reasons why some obese individuals fail to display cardiovascular risks, including hypertension.

Underlying Mechanisms

Many components of the BBS phenotype appear to involve defects in the central nervous system (CNS). This is supported by the observation that BBS genes and proteins are present throughout the brain and that BBS individuals and mice display cognitive, behavioral and neuroanatomical phenotypes.^{9, 63, 65–67} The relevance of the neurogenic mechanisms for the BBS phenotype is further demonstrated in mice carrying CNS-specific disruption of the BBSome. These mice recapitulate many features of BBS including the increase in blood pressure (Figure 2). Indeed, mice bearing disruption of the BBSome by deletion of the Bbs1 gene in the CNS develop obesity and hypertension.^{42, 68} This was associated with increased renal sympathetic outflow and exaggerated blood pressure decrease in response to ganglionic blockade, confirming that the increase in blood pressure in BBS is sympathetically mediated. These phenotypes were reproduced by disrupting the BBSome in neurons expressing the LepRb.

Figure 2:

Depiction of the cardiovascular effects evoked by deficiency in the BBSome and related proteins in keys organs (brain, kidney, heart and vasculature) involved in the control of various cardiovascular parameters. See the text for details (e.g., whether the renal effects are due to intrinsic or extrinsic factors).

The critical role of the BBSome in ciliary function led us to consider the possible contribution of cilia to the increase in blood pressure and sympathetic tone evoked by BBSome deficiency in the LepRb-expressing neurons. However, ablation of cilia through the deletion of the Ift88 gene in the LepRb neurons was associated with an increase in body weight and fat mass and a decrease in heart rate, but no significant change in blood pressure or renal sympathetic nerve traffic.⁶⁸ These findings indicate that the role of the BBSome in blood pressure and sympathetic control is not linked to its involvement in ciliary functions, pointing to other processes which remain to be determined.

Assessing the blood pressure effect of BBSome disruption in select hypothalamic neuronal populations such as those expressing AgRP (agouti-related protein), POMC (proopiomelanocortin) or SF1 (steroidogenic factor 1) yielded different outcomes than BBSome deficiency in the CNS or the LepRb neurons. Indeed, BBSome deficiency in AgRP or POMC neurons led to an increase in adiposity and sympathetic traffic to the kidney and splanchnic bed, but no change in blood pressure.⁶⁸ Disruption of the BBSome in SF1 neurons also caused obesity and splanchnic sympathetic nerve activation without significant change in blood pressure.⁶⁹ These findings show that an increase in renal and/or splanchnic sympathetic tone does not necessarily translate into blood pressure elevation. Counterregulatory mechanisms can offset the increase in sympathetic tone to maintain normal blood pressure. Consistent with such possibility, mice lacking the Bbs1 gene in POMC neurons exhibited impaired vascular contraction evoked by norepinephrine, indicative of adrenergic receptor desensitization. This is further supported by the decreased vascular α_1A- and α_1D-adrenergic receptors expression in mice lacking the Bbs1 gene in POMC or AgRP neurons but not in the LepRb neurons.

It should be noted that a similar phenomenon of vascular adrenergic receptor desensitization was described in obese normotensive subjects⁷⁰. This raises the interesting possibility that dysfunction of the BBSome in certain neuronal populations may contribute to the vascular adrenergic receptor desensitization and protection from hypertension in common obesity. This highlights the need for additional studies to assess the mechanisms underlying the regulation of blood pressure, sympathetic tone, and vascular reactivity by the neuronal BBSome.

BBSome AND RENAL DISEASES

Human Studies

Renal diseases are highly prevalent in BBS and an important cause of death.⁷¹ Up to 82% of BBS patients display some form of structural and/or functional abnormalities of the kidneys. Although highly variable among BBS patients, the renal phenotypes include cystic or dysplastic disease, decline in creatinine clearance, and other concentrating and filtrating defects (Figure 2).^{72, 73} These renal abnormalities, which often manifest very early in life, can progress to end-stage renal disease needing dialysis or transplantation. As with hypertension, renal impairments were found to be associated with all BBS genotypes except BBS2.⁶³ This finding points to renal defects as a potential cause of hypertension in BBS. Notably, patients carrying mutations in BBSome component genes display less severe renal phenotypes than those with mutations in the genes encoding BBS chaperonins.^{73, 74} For instance, patients with BBS10 gene mutations were found to be more likely to develop chronic kidney disease than those with BBS1 gene mutations.⁷⁴

Animal Studies

Analysis of the renal morphology of BBS mice revealed the existence of inflammatory infiltration, which was more pronounced in mice lacking the Bbs4 gene relative to Bbs2 gene.⁷⁵ Consistent with the presence of inflammation, BBS mice showed elevated renal expression of inducible nitric oxide synthase (iNOS) which was more pronounced in Bbs4-null mice. Conversely, endothelial nitric oxide synthase (eNOS) level was significantly decreased in the kidneys of Bbs4-, but not in Bbs2-, null mice. In addition to the enhanced inflammatory infiltration, Bbs4, but not Bbs2, knockout mice displayed significant glomerular changes including glomerular cysts. These morphological changes were associated with alterations in excretion of water and electrolytes as indicated by the decreased urine output and the increased concentration of sodium and blood urea nitrogen.⁷⁵

Underlying Mechanisms

Ciliary defects have been proposed as a potential cause of kidney disease in BBS. This is based on the observation that BBS knockout mice display shorter primary cilia in the renal epithelium.^{10, 36, 76} However, this is not a universal finding, as Bbs4 and Bbs10 knockout mice exhibit normal renal cilia.^{36, 77} Nonetheless, normal cilia appearance does not exclude ciliary dysfunction caused by mistraffificking of proteins such as PC1 and PC2 or aberrant ciliary signaling such as imbalanced Wnt pathway. Activation of mTOR (mechanistic target of rapamycin) signaling has also been suggested as a potential contributor to the development of cystic kidney disease in BBS based on a study that used rapamycin in zebrafish models.⁷⁸ Resistance to vasopressin resulting in the inability of this hormone to activate luminal aquaporin 2 in the collecting ducts is a putative mechanism proposed to explain the concentrating and excretory defects in BBS⁷⁹. In addition, a decrease in the renal expression of the transient receptor potential vanilloid (TRPV)1 and TRPV4 may contribute to BBS-associated renal dysfunction.⁷⁵

It is most noteworthy that elimination of obesity by calorie restriction rescued the morphological and molecular changes associated with BBS in mice.⁷⁵ This finding points to central mechanisms (e.g., renal sympathetic excitation) or systemic factors as the cause of renal abnormalities in BBS. This is further supported by the demonstration that in contrast to the global Bbs10 gene deletion, which causes various renal abnormalities, mice lacking the Bbs10 gene specifically in the renal epithelial cells displayed no morphological or molecular changes in the kidney.⁷⁷ It will be important to identify the extra renal factors underlying the kidney phenotypes of BBS that can be targeted to preserve renal function in syndromic patients.

BBSome AND VASCULATURE DYSFUNCTION

Human and Animal Studies

Although vascular functions have not been studied in BBS patients, the common occurrence in BBS individuals of hyperfibrigenemia and other vascular changes such as bilateral arterial occlusions and microaneurysms indicate vascular involvement of the BBSome (Figure 2). In line with this, genes encoding BBS proteins including components of the BBSome are expressed in endothelial and smooth muscle cells as well as vessels.⁸⁰

Study of BBS mouse models revealed that disruption of Bbs genes differentially affects vascular function.⁸⁰ Indeed, loss of the Bbs2, but not Bbs6, gene is associated with enhancement of the endothelium-dependent relaxation and attenuation of the contractile responses evoked by serotonin and endothelin-1. Moreover, Bbs2-null mice displayed an upregulation of expression of the nitric oxide–producing enzymes (eNOS and iNOS) and decreased expression of membrane subunits of NADPH oxidase (p22^phox and p47^phox) in the aorta. These vascular changes may contribute to the contrasting blood pressure effects caused by mutations in Bbs genes discussed above. It remains unclear, however, how disruption of two Bbs genes can yield such contrasting changes in vascular function. This raises the interesting possibility that the role of the product of these genes in vascular regulation is not linked to the BBSome, but this needs to be tested.

The generation of conditional knockout mice lacking the Bbs1 gene specifically in the smooth muscle or endothelial cells allowed a better appreciation of the role of the BBSome in vascular regulation without the cofounding effects of extrinsic factors. Smooth muscle–specific inducible deletion of the Bbs1 gene reduces relaxation and enhances contractility of vascular rings and increases aortic stiffness.⁵⁵ This was associated with an enhanced endothelin-1-induced contractility of mesenteric arteries. Deletion of the Bbs1 gene in endothelial cells also altered vascular function as indicated by the impaired acetylcholine-induced vasorelaxation in both the aorta and mesenteric artery.⁸¹ Additionally, we showed increased contractile response to activation of thromboxane A2 receptor in the mesenteric artery of endothelial cell Bbs1 conditional null mice. It should be noted that the vascular alterations evoked by smooth muscle and endothelial Bbs1 gene deletion were independent of changes in arterial blood pressure. Remarkably, the vascular dysfunction induced by disruption of the endothelial BBSome had ramifications on other physiological functions such as adiposity and hepatic and retinal functions.⁸¹

Underlying Mechanisms

Analysis of the mechanisms involved in the vascular defects induced by smooth muscle and endothelial cell Bbs1 gene deletion revealed an increase in vascular angiotensinogen gene expression, implicating the renin-angiotensin system in the vascular dysfunction associated with BBS. This was further confirmed by the ability of losartan to correct the enhanced endothelin-1-induced contractility of mesenteric artery of smooth muscle–specific Bbs1 knockout mice. Together, these findings highlight the importance of the smooth muscle and endothelial BBSome in the control of vascular function through modulation of the renin-angiotensin system. Future studies of the role of the BBSome will elucidate the molecular mechanisms that underlie vascular diseases and identify novel therapeutic targets for the clinical treatment of vascular dysfunction, a major cardiovascular risk.

BBSome AND CARDIAC DEFECTS

Human Studies

A high incidence of congenital or acquired cardiac abnormalities has been associated with BBS. Analysis of 50 BBS patients of three inbred Bedouin families revealed that 50% of the cases display some congenital heart defects such as hypertrophy of interventricular septum and dilated cardiomyopathy.⁸² Other cardiac defects noted in BBS patients include aortic valve stenosis, coarctation of the aorta, and abnormal ejection fraction (Figure 2).^{63, 73} These findings have led to the suggestion that echocardiographic examination should be included in the clinical evaluation and follow-up of BBS patients. It should be noted, however, that some of the cardiac changes associated with BBS such as the acquired heart disease may be secondary to other conditions such as hypertension and renal abnormalities.

Animal Studies

To determine whether BBS mice recapitulate the cardiac defects observed in BBS patients, an echocardiography study was carried out in three mouse models (Bbs2^−/−, Bbs4^−/−, and Bbs6^−/−).⁸ However, there was no difference in BBS mice compared to wild type animals for any of the cardiac parameters measured. To assess whether cardiac abnormalities may be slow in onset and develop at later stages, echocardiography was performed in older (6–12 month) BBS mice, but no significant difference in the echocardiography data was observed between older BBS mice relative to the littermate controls.⁸ The lack of cardiac dysfunction in BBS mice indicate that BBSome deficiency may protect against the cardiac side effects of obesity and hypertension. However, the non-recapitulation of the cardiac phenotypes in mouse models has hampered the dissection of the molecular bases of the heart defects observed in BBS patients.

Underlying Mechanisms

A study using zebrafish provided some insight into the mechanisms explaining how loss of BBS proteins lead to congenital heart defects.⁵⁴ This involves BBS6 modulation of SWI/SNF chromatin remodeling complex discussed above, which has been linked to congenital heart defects. Interestingly, overexpression of BBS6 was found to cause differential expression of 449 genes in the heart.⁵⁴ This includes over 15 genes with a known role in heart development and cardiac function such as secondary heart field development, cardiac valve formation, and heart morphogenesis. Moreover, comparing the list of differentially expressed genes following BBS6 overexpression with predicted human homologs and filtered against the list of genes in which de novo mutations were identified in patients with congenital heart disease resulted in an overlap of 30 genes.⁵⁴ These findings point to a potential role for BBS6 and perhaps other BBS proteins in the development of heart disease in non-BBS patients.

CONCLUSIONS AND PERSPECTIVES

The high prevalence of cardiovascular risk factors in BBS, a rare monogenic human condition, has triggered a tremendous interest in deciphering the underlying molecular, cellular, and physiological mechanisms. This has led to the identification of the BBSome and related proteins as important players in cardiovascular regulation, including blood pressure, renal function, and vascular reactivity. Substantial progress has also been made in understanding the pathophysiological processes causing BBS-associated cardiovascular risks. However, this progress has not translated into the clinic. For instance, there is currently no recommendation as how to manage the various cardiovascular risks associated with this unique group of individuals. This is urgently needed given that cardiovascular diseases are the main cause of death of BBS patients.

There is also a need for additional studies to better understand the role of the BBSome in the control of various cellular processes, including those involved in cardiovascular health and disease. This includes deciphering the contribution of the BBSome versus the subcomplexes and individual BBS proteins to the regulation of different cellular functions and fundamental molecular mechanisms such as the renin-angiotensin system. This will be necessary to decipher the described link between BBS genes and different cardiovascular risks, including hypertension in non-BBS individuals.

In addition to the cardiovascular risks discussed above, a number of other abnormalities have been described in BBS patients; these include dyslipidemia (increase in the LDL cholesterol and triglyceride and a decrease in HDL cholesterol) and elevated levels of C-reactive protein and of C peptide. These risk factors may be linked to the high prevalence of obesity, particularly visceral obesity, in BBS. However, this remains to be determined. For instance, in contrast to common human obesity, type 2 diabetes associated with BBS was found to be caused by intrinsic insulin resistance due to the mishandling of the insulin receptor.⁴⁴ Thus, it is possible that these other cardiovascular risks develop in a manner independent of obesity. Uncovering the molecular causes could lead to the identification of novel mechanisms of cardiovascular disease.