Elevated circulating metalloproteinase 7 predicts recurrent cardiovascular events in patients with carotid stenosis: a prospective cohort study

Background: Major adverse cardiovascular events are the main cause of morbidity and mortality over the long term in patients undergoing carotid endarterectomy. There are few reports assessing the prognostic value of markers of inflammation in relation to the risk of cardiovascular disease after carotid endarterectomy. Here, we aimed to determine whether matrix metalloproteinases (MMP-1, MMP-2, MMP-7, MMP-9 and MMP-10), tissue inhibitor of MMPs (TIMP-1) and in vivo inflammation studied by 18F-FDG-PET/CT predict recurrent cardiovascular events in patients with carotid stenosis who underwent endarterectomy. Methods: This prospective cohort study was carried out on 31 consecutive patients with symptomatic (23/31) or asymptomatic (8/31) severe (> 70%) carotid stenosis who were scheduled for carotid endarterectomy between July 2013 and March 2016. In addition, 26 healthy controls were included in the study. Plasma and serum samples were collected 2 days prior to surgery and tested for MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, TIMP-1, high-density lipoprotein, low-density lipoprotein, high-sensitivity C-reactive protein and erythrocyte sedimentation rate. 18F-FDGPET/CT focusing on several territories’ vascular wall metabolism was performed on 29 of the patients because of no presurgical availability in 2 symptomatic patients. Histological and immunohistochemical studies were performed with antibodies targeting MMP-10, MMP-9, TIMP-1 and CD68. Results: The patients with carotid stenosis had significantly more circulating MMP-1, MMP-7 and MMP-10 than the healthy controls. Intraplaque TIMP-1 was correlated with its plasma level (r = 0.42 P = .02) and with 18F-FDG uptake (r = 0.38 P = .05). We did not find any correlation between circulating MMPs and in vivo carotid plaque metabolism assessed by 18F-FDG-PET. After a median follow-up of 1077 days, 4 cerebrovascular, 7 cardiovascular and 11 peripheral vascular events requiring hospitalization were registered. Circulating MMP-7 was capable of predicting events over and above the traditional risk factors (HR = 1.15 P = .006). When the model was associated with the variables of interest, the risk predicted by 18F-FDG-PET was not significant. Conclusions: Circulating MMP-7 may represent a novel marker for recurrent cardiovascular events in patients with moderate to severe carotid stenosis. MMP-7 may reflect the atherosclerotic burden but not plaque inflammation in this specific vascular territory

Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants

The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF

Stylohyoid Complex (Eagle) Syndrome Starting in a 9-Year-Old Boy

Background There are only four previous pediatric reports of the glossopharyngeal neuralgic form of the stylohyoid complex syndrome. Stylohyoid complex has merely been described as cases of glossopharyngeal neuralgia in children. Case Report A 12-year-old boy came to our hospital because of recurrent episodes of severe cranial pain (9/10) lasting for 5 to 15 minutes. Pain affected the right tonsillar fossa, ear, and mastoid region. Since the start at the age of 9 years, the frequency of painful episodes has progressively increased: when admitted to our clinics 3 years later, the child was having up to five episodes daily in spite of analgesic, antiepileptic, and antidepressant drugs; he had abandoned school and leisure. Between episodes, neurological examination detected only discomfort to pressure on the right tonsillar fossa. Three-dimensional computed tomography images of the skull base showed an elongated right styloid process and bilateral calcification of the stylohyoid ligament. After surgical excision of the right styloid process and of part of the stylohyoid ligament, the glossopharyngeal painful episodes ceased. The patient remains asymptomatic seven years later. Conclusion In spite of its rarity in childhood, this debilitating but treatable syndrome should be kept in mind for the differential diagnosis of recurrent cranial pain in the pediatric population

Systemic and cerebral iron homeostasis in ferritin knock-out mice.

Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect

Elevated circulating metalloproteinase 7 predicts recurrent cardiovascular events in patients with carotid stenosis: a prospective cohort study

Background: Major adverse cardiovascular events are the main cause of morbidity and mortality over the long term in patients undergoing carotid endarterectomy. There are few reports assessing the prognostic value of markers of inflammation in relation to the risk of cardiovascular disease after carotid endarterectomy. Here, we aimed to determine whether matrix metalloproteinases (MMP-1, MMP-2, MMP-7, MMP-9 and MMP-10), tissue inhibitor of MMPs (TIMP-1) and in vivo inflammation studied by 18F-FDG-PET/CT predict recurrent cardiovascular events in patients with carotid stenosis who underwent endarterectomy. Methods: This prospective cohort study was carried out on 31 consecutive patients with symptomatic (23/31) or asymptomatic (8/31) severe (> 70%) carotid stenosis who were scheduled for carotid endarterectomy between July 2013 and March 2016. In addition, 26 healthy controls were included in the study. Plasma and serum samples were collected 2 days prior to surgery and tested for MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, TIMP-1, high-density lipoprotein, low-density lipoprotein, high-sensitivity C-reactive protein and erythrocyte sedimentation rate. 18F-FDGPET/CT focusing on several territories’ vascular wall metabolism was performed on 29 of the patients because of no presurgical availability in 2 symptomatic patients. Histological and immunohistochemical studies were performed with antibodies targeting MMP-10, MMP-9, TIMP-1 and CD68. Results: The patients with carotid stenosis had significantly more circulating MMP-1, MMP-7 and MMP-10 than the healthy controls. Intraplaque TIMP-1 was correlated with its plasma level (r = 0.42 P = .02) and with 18F-FDG uptake (r = 0.38 P = .05). We did not find any correlation between circulating MMPs and in vivo carotid plaque metabolism assessed by 18F-FDG-PET. After a median follow-up of 1077 days, 4 cerebrovascular, 7 cardiovascular and 11 peripheral vascular events requiring hospitalization were registered. Circulating MMP-7 was capable of predicting events over and above the traditional risk factors (HR = 1.15 P = .006). When the model was associated with the variables of interest, the risk predicted by 18F-FDG-PET was not significant. Conclusions: Circulating MMP-7 may represent a novel marker for recurrent cardiovascular events in patients with moderate to severe carotid stenosis. MMP-7 may reflect the atherosclerotic burden but not plaque inflammation in this specific vascular territory

Strategy employed for the inactivation of the murine ferritin light and heavy genes.

<p>For the inactivation of the <i>Ftl</i> gene, exons 1 and 2 were deleted (A). The deletion targeting vector contained the <i>MC1tk</i> (thymidine kinase) expression cassette, 5.72 kb of 5’ <i>Ftl</i> homology, a <i>pGK-neo</i> (neomycin-resistance gene) expression cassette, 0.83 kb of the <i>Ftl</i> gene containing exons 1 and 2, and 5.74 kb of 3’ <i>Ftl</i> homology. Three loxP sites were introduced by targeted mutagenesis between the <i>neo</i> cassette and after exon 2 of <i>Ftl</i>. Homologous recombination in ES cells identified by Southern blot hybridization analysis using the external probe A after digestion with XhoI. The wild-type allele has 8.03 kb and the floxed allele has 7.30 kb (B). Germline chimeras were crossed with C57Bl/6 females, and mice carrying the floxed <i>Ftl</i> (<i>Ftl</i>^<i>f/+</i>) allele were then crossed with mice expressing the <i>Cre</i> recombinase. Mice showing the deletion of exons 1 and 2 of <i>Ftl</i> and the <i>neo</i> cassette were crossed with C57Bl/6 mice to remove the <i>Cre</i> allele. Mice were maintained in C57Bl/6 background and the presence of the <i>Ftl</i> allele determined by PCR, resulting in a 180-bp fragment for the wild-type allele and in a 204-bp fragment for the knock-out allele (B). The four exons of the mouse <i>Fth</i> gene were targeted for deletion (C). The deletion targeting vector contained the <i>MC1tk</i> expression cassette, 4.44 kb of 5’ <i>Fth</i> homology, a <i>pGK-neo</i> expression cassette, 2.4 kb of the <i>Fth</i> gene containing all four exons, and 5.0 kb of 3’ <i>Fth</i> homology. Three loxP sites were introduced by targeted mutagenesis between the neo cassette and after exon 4 of <i>Fth</i>. Homologous recombination in ES cells identified by Southern blot hybridization analysis using the external probe A after digestion with HindIII. The wild-type allele has 6.25 kb and the floxed <i>Fth</i> (<i>Fth</i>^<i>f/+</i>) allele has 5.62 kb (D). Germline chimeras were crossed with C57Bl/6 females, and mice carrying the floxed <i>Fth</i> allele were then crossed with mice expressing the <i>Cre</i> recombinase. Mice showing complete loss of <i>Fth</i> and the <i>neo</i> cassette were crossed with C57Bl/6 mice to remove the <i>Cre</i> allele. Mice were maintained in C57Bl/6 background and the presence of the <i>Fth</i> allele determined by PCR, resulting in a 252-bp fragment for the wild-type allele and in a 354-bp fragment for the knock-out allele (D). The symbol ► represents the loxP sites; f, floxed alleles.</p

Deletion of one or both alleles of the <i>Ftl</i> gene causes a mild increase of serum iron levels that was significant for Ftl^-/- mice (<i>p</i> < 0.05) (A), and a decrease of UIBC levels in Ftl^-/- mice that did not reach statistical significance (<i>p</i> < 0.06) (B).

<p>Wild-type control (+/+; n = 12), <i>Ftl</i>^+/- (+/-; n = 8), and <i>Ftl</i>^-/- (-/-; n = 10) mice were analyzed. Samples were analyzed by a two-tailed <i>t</i>-test and results considered significant for <i>p</i> < 0.05.</p