Ilaria Gatto

Single nucleotide polymorphisms associated with sporadic brain arteriovenous malformations: where do we stand?

Brain arteriovenous malformations are characterized by a tangle of abnormal vessels directly shunting blood from the arterial to venous circulation. They are known to occur either sporadically or in the context of well-defined genetic disorders. Haemorrhage represents the most severe clinical manifestation, whereas other common symptoms include headache, seizures and neurological deficits. Although sporadic forms do not recognize a specific genetic cause, in recent years, it has been hypothesized that genes involved in angiogenesis and inflammation or coding for proteins, such as fibronectins, laminins and integrins, may play a role in the pathophysiology of brain arteriovenous malformations. More recently, a new trend of genetic studies has investigated the association between sporadic arteriovenous malformations and single nucleotide polymorphisms, single base variations between genomes within members of a biological species or between paired chromosomes in an individual, which may determine the susceptibility to develop complex diseases and influence their natural history. Several polymorphisms in two different families of genes have been associated with disease susceptibly and increased haemorrhagic risk. These genes are mainly involved in the inflammatory cascade and in the regulation of angiogenesis. However, most of the investigated polymorphisms have been selected on the basis of candidate genes because of their potential functional role in the pathogenesis of brain arteriovenous malformations or in other cerebrovascular diseases. Only one hypothesis-free genome-wide association study in a small number of patients has been performed so far, but it was unable to identify significant associations between brain arteriovenous malformations and specific genetic loci. In this article, we review and analyse the polymorphisms investigated to date in association with sporadic brain arteriovenous malformations in the medical literature. We discuss the biological, pathophysiological and clinical implications of these studies, with particular attention to the prediction of haemorrhagic risk and the possibility of building genetic profiles capable of defining the architectural features of the malformations and predict their evolution and natural history. We also present a joint analysis of the risk estimates found by the studies in literature that have evaluated the association between single nucleotide polymorphisms and brain arteriovenous malformation susceptibility and risk of bleeding. This analysis shows a statistically significant association between the interleukin 6 -174G>C (odds ratio = 1.97; 95% confidence interval: 1.15-3.38) and the tumour necrosis factor α -238G>A (odds ratio = 2.19; 95% confidence interval: 1.25-3.83) gene polymorphisms and risk of intracranial haemorrhage and between the activin-like kinase 1 (also known as ACVRL1) intervening sequence 3 -35A>G (odds ratio = 2.42; 95% confidence interval: 1.54-3.8) gene polymorphism and disease susceptibility.

Pleiotropic Beneficial Effects of Sonic Hedgehog Gene Therapy in an Experimental Model of Peripheral Limb Ischemia

We have previously shown that the signaling pathway of the embryonic morphogen Sonic hedgehog (Shh) is recapitulated in the postnatal skeletal muscle in response to ischemia. We have also demonstrated that Shh is an indirect angiogenic agent upregulating various families of angiogenic growth factors and that Shh gene therapy improves angiogenesis and heart function in experimental models of myocardial ischemia. Based on these findings, we hypothesized that Shh gene therapy is beneficial in an experimental model of peripheral ischemia. We found that intramuscular (i.m.) treatment with a plasmid encoding the Shh human gene (phShh) increased blood flow, capillary density, and arteriole density in mice in which peripheral circulation of the hindlimb was disrupted by removal of the common femoral artery. Shh gene therapy also enhanced vasculogenesis, by increasing the number of circulating bone marrow (BM)-derived endothelial precursors and improving the contribution of these cells to the process of neovascularization. Finally, phShh treatment induced upregulation of prototypical angiogenic, arteriogenic, and vasculogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and stromal cell-derived factor-1 (SDF-1α). These data suggest that Shh gene therapy merits further investigation for its ability to trigger the expression of potent trophic factors and stimulate pleiotropic aspects of neovascularization in the setting of ischemia.

Angiogenic Impairment of the Vascular Endothelium A Novel Mechanism and Potential Therapeutic Target in Muscular Dystrophy

Objective— Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results— We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx -derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx -derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions— These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy. # Significance {#article-title-36}Objective—Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results—We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated &bgr;-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions—These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Combined Therapy with Sonic Hedgehog Gene Transfer and Bone Marrow-Derived Endothelial Progenitor Cells Enhances Angiogenesis and Myogenesis in the Ischemic Skeletal Muscle

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

Angiogenic Impairment of the Vascular Endothelium

Objective— Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results— We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx -derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx -derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions— These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy. # Significance {#article-title-36}Objective—Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results—We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated &bgr;-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions—These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Sonic hedgehog gene therapy increases the ability of the dystrophic skeletal muscle to regenerate after injury

The Hedgehog (Hh) pathway is a crucial regulator of muscle development during embryogenesis. We have previously demonstrated that Sonic hedgehog (Shh) regulates postnatal myogenesis in the adult skeletal muscle both directly, by acting on muscle satellite cells, and indirectly, by promoting the production of growth factors from interstitial fibroblasts. Here, we show that in mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, progression of the dystrophic pathology corresponds to progressive inhibition of the Hh signaling pathway in the skeletal muscle. We also show that the upregulation of the Hh pathway in response to injury and during regeneration is significantly impaired in mdx muscle. Shh treatment increases the proliferative potential of satellite cells isolated from the muscles of mdx mice. This treatment also increases the production of proregenerative factors, such as insulin-like growth factor-1 and vascular endothelial growth factor, from fibroblasts isolated from the muscle of mdx mice. In vivo, overexpression of the Hh pathway using a plasmid encoding the human Shh gene promotes successful regeneration after injury in terms of increased number of proliferating myogenic cells and newly formed myofibers, as well as enhanced vascularization and decreased fibrosis.

Association between the rs1333040 polymorphism on the chromosomal 9p21 locus and sporadic brain arteriovenous malformations.

Background Single nucleotide polymorphisms (SNPs) on chromosome 9p21 have been recently associated with intracranial aneurysms and stroke. In this study, we tested the association between the rs1333040C>T polymorphism on the 9p21 locus and sporadic brain arteriovenous malformations (BAVMs). Methods We studied 78 patients with sporadic BAVMs and 103 unaffected controls. Genomic DNA was isolated from peripheral blood and the rs1333040C>T polymorphism was assessed by PCR–restriction fragment length polymorphism using the BsmI restriction endonuclease. Results We found that the distribution of the three genotypes (TT/TC/CC) of the rs1333040 polymorphism was significantly different between cases and controls (p=0.02). Using dominant, recessive and additive genetic models, we found that the TT genotype and the T allele were significantly more common in the BAVM group than in controls. We also evaluated whether the rs1333040 polymorphism was associated with prototypical angio-architectural features of BAVMs (such as nidus size, venous drainage pattern and Spetzler–Martin grading) and with the occurrence of seizures and bleeding. We detected a significant association between the homozygous T allele in the recessive model and BAVMs with a nidus >4 cm in diameter. Deep venous drainage was significantly more frequent among subjects carrying at least one T allele in the dominant model. Patients with seizures showed a significant association with the TT genotype and the T allele in all genetic models examined whereas those who experienced intracranial bleeding showed a significant association with the T allele in the trend model. Conclusions This is the first study demonstrating an association between an SNP of the 9p21 region and sporadic BAVMs. Our results emphasise the relevance of this chromosomal locus as a common risk factor for various forms of cerebrovascular diseases.

Sonic Hedgehog Therapy in a Mouse Model of Age-Associated Impairment of Skeletal Muscle Regeneration

Sonic hedgehog (Shh) is a morphogen regulating muscle development during embryogenesis. We have shown that the Shh pathway is postnatally recapitulated after injury and during regeneration of the adult skeletal muscle and regulates angiogenesis and myogenesis after muscle injury. Here, we demonstrate that in 18-month-old mice, there is a significant impairment of the upregulation of the Shh pathway that physiologically occurs in the young skeletal muscle after injury. Such impairment is even more pronounced in 24-month-old mice. In old animals, intramuscular therapy with a plasmid encoding the human Shh gene increases the regenerative capacities of the injured muscle, in terms of Myf5-positive cells, regenerating myofibers, and fibrosis. At the molecular level, Shh treatment increases the upregulation of the prototypical growth factors, insulin-like growth factor-1 and vascular endothelial growth factor. These data demonstrate that Shh increases regeneration after injury in the muscle of 24-month-old mice and suggest that the manipulation of the Shh pathway may be useful for the treatment of muscular diseases associated with aging.

Association between Polymorphisms rs1333040 and rs7865618 of Chromosome 9p21 and Sporadic Brain Arteriovenous Malformations

Background: The chromosomal locus 9p21 is a novel genetic marker for a variety of cardiovascular and cerebrovascular diseases. In a recent study, we have demonstrated an association between the single nucleotide polymorphism (SNP) rs1333040C>T on chromosome 9p21 and sporadic brain arteriovenous malformations (BAVMs). Here, we extended our analysis to an additional SNP on chromosome 9p21 (rs7865618A>G) and increased our sample size including BAVMs from two different Italian neurosurgical centers. Methods: We studied 206 patients with sporadic BAVMs and 171 unaffected controls. Genomic DNA was isolated from peripheral blood and the rs1333040C>T and rs7865618A>G polymorphisms were assessed by PCR-RFLP using the BsmI and MspI restriction endonucleases, respectively. For each SNP, we performed dominant, recessive, and additive genetic models. Results: The distribution of the three possible genotypes of rs1333040 (TT, TC and CC) was statistically different between cases and controls (p = 0.0008). The TT genotype was significantly associated with BAVMs both in the dominant (p = 0.013) and recessive (p = 0.012) models. The T allele was significantly associated with BAVMs in the additive model (p = 0.002). Also the distribution of the three possible genotypes of rs7865618 (GG, AG and AA) was statistically different between cases and controls (p = 0.005), and the GG genotype and G allele were significantly associated with BAVMs in the dominant (p = 0.032), recessive (p = 0.007), and additive models (p = 0.009). We also detected a significant association between BAVMs with large nidus size and the GG genotype and G allele of rs7865618 and the TT genotype of rs1333040. A deep venous drainage was instead associated with the TT genotype of the rs1333040 and the GG genotype of the rs7865618. The occurrence of bleeding was associated with the TT genotype and T allele of rs1333040, while the presence of seizures appeared associated with the GG genotype of rs7865618. Conclusions: SNPs of the 9p21 region, in addition to be genetic markers for coronary artery disease, stroke, and intracranial aneurysms, are associated with sporadic BAVMs. These results extend and strengthen the role of the 9p21 chromosomal region as a common risk factor for cerebrovascular diseases.

HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection

Circulating microparticles (MPs) are novel potential biomarkers in cancer patients. Their role in hepatocellular carcinoma (HCC) is under intensive investigation. In this study, we tested the hypothesis that MPs expressing the antigen HepPar1 are increased in the blood of subjects with HCC and may serve as markers of early recurrence after liver resection (LR). We studied 15 patients affected by HCC undergoing LR, and used flow cytometry to assess the number of circulating HepPar1+ MPs. Ten subjects without HCC (five with liver cirrhosis and five with healthy livers) were used as controls. After LR, HCC patients underwent a follow-up to check for early recurrence, which occurred in seven cases. The number of circulating HepPar1+ MPs was significantly higher in subjects affected by HCC, compared to individuals without cancer (p < 0.01). We also found that, among HCC patients, the number of circulating HepPar1+ MPs, measured before LR, was significantly higher in those who displayed early recurrence compared to those without recurrence (p = 0.02). Of note, other types of circulating MPs, such as those derived from endothelial cells (CD144+) or those produced by the activated endothelium (CD144+/CD62+), were not associated with HCC, nor could they predict HCC recurrence. HepPar1+ MPs deserve further investigation as novel biomarkers of disease and prognosis in HCC patients.