Igor Giarretta

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.

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.

The 9p21 Rs 1333040 polymorphism is associated with coronary microvascular obstruction in ST-segment elevation myocardial infarction treated by primary angioplasty

Background: Microvascular obstruction (MVO) after primary percutaneous coronary intervention (pPCI) leads to higher incidence of both early and late complications. A number of single nucleotide polymorphisms in 9p21 chromosome have been shown to affect angiogenesis in response to ischaemia. In particular, Rs1333040 with its three genotypic vriants C/C, T/C and T/T might influence the occurrence of MVO after pPCI. Methods: We enrolled ST-elevation myocardial infarction (STEMI) patients undergoing pPCI. The Rs1333040 polymorphism was evaluated by polymerase chain reaction-restriction fragment length polymorphism using restriction endonucleases (Bsml). Two expert operators unaware of the patients’ identity performed the angiographic analysis; collaterals were assessed applying Rentrop’s classification. Angiographic MVO was defined as a post-pPCI Thrombolysis In Myocardial Infarction (TIMI)<3 or TIMI 3 with myocardial blush grade 0 or 1, whereas electrocardiographic MVO was defined as ST segment resolution <70% one hour after pPCI. Results: Among our 133 STEMI patients (mean age 63 ± 11 years, men 72%), 35 (26%) and 53 (40%) respectively experienced angiographic or electrocardiographic MVO. Angiographic and electrocardiographic MVO were different among the three variants (p= 0.03 and p=0.02 respectively). In particular, T/T genotype was associated with a higher incidence of both angiographic and electrocardiographic MVO compared with C/C genotype (p=0.04 and p=0.03 respectively). Moreover, Rentrop score <2 detection rate differed among the three genotypes (p=0.03). In particular T/T genotype was associated with a higher incidence of a Rentrop score <2 as compared with C/C genotype (p= 0.02). Conclusion: Rs1333040 polymorphism genetic variants portend different MVO incidence. In particular, T/T genotype is related to angiographic and electrocardiographic MVO and to worse collaterals towards the culprit artery.

The Hedgehog Signaling Pathway in the Ischemic Heart, Brain, and Skeletal Muscle

Hedgehog (Hh) proteins are prototypical morphogens known to regulate epithelial/mesenchymal interactions during embryonic development. In addition to its pivotal role in embryogenesis, the Hh signaling pathway may be recapitulated in post-natal life in a number of physiological and pathological conditions, including ischemia. This review highlights the involvement of Hh signaling in ischemic tissue regeneration and angiogenesis, with particular attention to the heart, the brain, and the skeletal muscle. Updated information on the potential role of the Hh pathway as a therapeutic target in ischemic condition is also presented.

Microparticles Produced by Activated Platelets Carry a Potent and Functionally Active Angiogenic Signal in Subjects with Crohn’s Disease

Microparticles (MPs) are submicron vesicles shed from various cell types upon activation, stimulation, and death. Activated platelets are an important source of circulating MPs in subjects with inflammatory diseases, including Crohn’s disease (CD). Angiogenesis is a hallmark of inflammation in CD and plays an active role in sustaining disease progression, while targeting angiogenesis may be an effective approach to block colitis. In this study, we analyzed the angiogenic content of the MPs produced by activated platelets in subjects with CD. We also evaluated whether the angiogenic signal carried by these MPs was functionally active, or able to induce angiogenesis. We found that, in subjects with CD, MPs produced by activated platelets contain significantly higher levels of angiogenic mRNAs, such as epidermal growth factor (EGF), platelet-derived growth factor-α (PDGFα), fibroblast growth factor (FGF-2), and angiopoietin-1 (ANGPT1), compared to MPs isolated from control subjects. They also contain significantly higher levels of prototypical angiogenic proteins, including vascular endothelial growth factor (VEGF), angiopoietin-1, endoglin, endothelin-1, pentraxin 3, platelet factor-4, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), and thrombospondin 1. The protein content of these MPs is functionally active, since it has the ability to induce a robust angiogenic process in an endothelial cell/interstitial cell co-culture in vitro assay. Our results reveal a potential novel mechanism through which the angiogenic signal is delivered in subjects with CD, with potentially important clinical and therapeutic implications.