CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

The role of cyclase-associated protein (CAP) in actin dynamics during cell motility and morphogenesis

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

Postmenopausal hot flushes, vascular health and hormone therapy

Vasomotor hot flushes are complained of by approximately 75% of postmenopausal women, but their frequency and severity show great individual variation. Hot flushes have been present in women attending observational studies showing cardiovascular benefit associated with hormone therapy use, whereas they have been absent or very mild in randomized hormone therapy trials showing cardiovascular harm. Therefore, if hot flushes are a factor connected with vascular health, they could perhaps be one explanation for the divergence of cardiovascular data in observational versus randomized studies. For the present study 150 healthy, recently postmenopausal women showing a large variation in hot flushes were studied in regard to cardiovascular health by way of pulse wave analysis, ambulatory blood pressure and several biochemical vascular markers. In addition, the possible impact of hot flushes on outcomes of hormone therapy was studied. This study shows that women with severe hot flushes exhibit a greater vasodilatory reactivity as assessed by pulse wave analysis than do women without vasomotor symptoms. This can be seen as a hot flush-related vascular benefit. Although severe night-time hot flushes seem to be accompanied by transient increases in blood pressure and heart rate, the diurnal blood pressure and heart rate profiles show no significant differences between women without and with mild, moderate or severe hot flushes. The levels of vascular markers, such as lipids, lipoproteins, C-reactive protein and sex hormone-binding globulin show no association with hot flush status. In the 6-month hormone therapy trial the women were classified as having either tolerable or intolerable hot flushes. These groups were treated in a randomized order with transdermal estradiol gel, oral estradiol alone or in combination with medroxyprogesterone acetate, or with placebo. In women with only tolerable hot flushes, oral estradiol leads to a reduced vasodilatory response and increases in 24-hour and daytime blood pressures as compared to women with intolerable hot flushes receiving the same therapy. No such effects were observed with the other treatment regimes or in women with intolerable hot flushes. The responses of vascular biomarkers to hormone therapy are unaffected by hot flush status. In conclusion, hot flush status contributes to cardiovascular health before and during hormone therapy. Severe hot flushes are associated with an increased vasodilatory, and thus, a beneficial vascular status. Oral estradiol leads to vasoconstrictive changes and increases in blood pressure, and thus to possible vascular harm, but only in women whose hot flushes are so mild that they would probably not lead to the initiation of hormone therapy in clinical practice. Healthy, recently postmenopausal women with moderate to severe hot flushes should be given the opportunity to use hormone therapy alleviate hot flushes, and if estrogen is prescribed for indications other than for the control of hot flushes, transdermal route of administration should be favored.

Regulation of Kinin receptor Expression in Heart Failure with a Focus on Vascular Endothelium

Accumulating evidence show that kinins, notably bradykinin (BK) and kallidin, have cardioprotective effects. To these include reduction of left ventricular hypertrophy (LVH) and progression of heart failure. The effects are mediated through two G protein-coupled receptors- bradykinin type-2 receptor (BK-2R) and bradykinin type -1 receptor (BK-1R). The widely accepted cardioprotective effects of BK-receptors relate to triggering the production and release of vasodilating nitric oxide (NO) by endothelial cells. They also exert anti-proliferative effects on fibroblasts and anti-hypertrophic effects on myocytes, and thus may play an essential role in the cardioprotective response to myocardial injury. The role for BK-1Rs in HF is based on experimental animal models, where the receptors have been linked to cardioprotective- but also to cardiotoxic -effects. The BK-1Rs are induced under inflammatory and ischemic conditions, shown in animal models; no previous reports, concerning BK-1Rs in human heart failure, have been presented. The expression of BK-2Rs is down-regulated in human end-stage heart failure. Present results showed that, in these patients, the BK-1Rs were up-regulated, suggesting that also BK-1Rs are involved in the pathogenesis of human heart failure. The receptors were localized mainly in the endothelium of intramyocardial coronary vessels, and correlated with the increased TNF-α expression in the myocardial coronary vessels. Moreover, in cultured endothelial cells, TNF-α was a potent trigger of BK-1Rs. These results suggest that cytokines may be responsible for the up-regulation of BK-1Rs in human heart failure. A linear relationship between BK-2R mRNA and protein expression in normal and failing human left ventricles implies that the BK-2Rs are regulated on the transcriptional level, at least in human myocardium. The expression of BK-2Rs correlated positively with age in normal and dilated hearts (IDC). The results suggest that human hearts adapts to age-related changes, by up-regulating the expression of cardioprotective BK-2Rs. Also, in the BK-2R promoter polymorphism -58 T/C, the C-allele was accumulated in cardiomyopathy patients which may partially explain the reduced number of BK-2Rs. Statins reduce the level of plasma cholesterol, but also exert several non-cholesterol-dependent effects. These effects were studied in human coronary arterial endothelial cells (hCAEC) and incubation with lovastatin induced both BK-1 and BK-2Rs in a time and concentration-dependent way. The induced BK-2Rs were functionally active, thus NO production and cGMP signaling was increased. Induction was abrogated by mevalonate, a direct HMG-CoA metabolite. Lovastatin is known to inhibit Rho activation, and by a selective RhoA kinase inhibitor (Y27632), a similar induction of BK-2R expression as with lovastatin. Interestingly a COX-2-inhibitor (NS398) inhibited this lovastatin-induction of BK-2Rs, suggesting that COX-2 inhibitors may affect the endothelial BK-2Rs, in a negative fashion. Hypoxia is a common denominator in HF but also in other cardiovascular diseases. An induction of BK-2Rs in mild hypoxic conditions was shown in cultured hCAECs, which was abolished by a specific BK-2R inhibitor Icatibant. These receptors were functionally active, thus BK increased and Icatibant inhibited the production of NO. In rat myocardium the expression of BK-2R was increased in the endothelium of vessels, forming at the border zone, between the scar tissue and the healthy myocardium. Moreover, in in vitro wound-healing assay, endothelial cells were cultured under hypoxic conditions and BK significantly increased the migration of these cells and as Icatibant inhibited it. These results show, that mild hypoxia triggers a temporal expression of functionally active BK-2Rs in human and rat endothelial cells, supporting a role for BK-2Rs, in hypoxia induced angiogenesis. Our and previous results show, that BK-Rs have an impact on the cardiovascular diseases. In humans, at the end stage of heart failure, the BK-2Rs are down-regulated and BK-1Rs induced. Whether the up-regulation of BK-1Rs, is a compensatory mechanism against the down-regulation of BK-2Rs, or merely reflects the end point of heart failure, remains to bee seen. In a clinical point of view, the up-regulation of BK-2Rs, under hypoxic conditions or statin treatment, suggests that, the induction of BK-2Rs is protective in cardiovascular pathologies and those treatments activating BK-2Rs, might give additional tools in treating heart failure.