Paradoxical aortic stiffening and subsequent cardiac dysfunction in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare disorder with devastating sequelae resulting in early death, presently thought to stem primarily from cardiovascular events. We analyse novel longitudinal cardiovascular data from a mouse model of HGPS (LmnaG609G/G609G) using allometric scaling, biomechanical phenotyping, and advanced computational modelling and show that late-stage diastolic dysfunction, with preserved systolic function, emerges with an increase in the pulse wave velocity and an associated loss of aortic function, independent of sex. Specifically, there is a dramatic late-stage loss of smooth muscle function and cells and an excessive accumulation of proteoglycans along the aorta, which result in a loss of biomechanical function (contractility and elastic energy storage) and a marked structural stiffening despite a distinctly low intrinsic material stiffness that is consistent with the lack of functional lamin A. Importantly, the vascular function appears to arise normally from the low-stress environment of development, only to succumb progressively to pressure-related effects of the lamin A mutation and become extreme in the peri-morbid period. Because the dramatic life-threatening aortic phenotype manifests during the last third of life there may be a therapeutic window in maturity that could alleviate concerns with therapies administered during early periods of arterial development.

Genetic and pharmacologic modulation of cementogenesis via pyrophosphate regulators.

Pyrophosphate (PPi) serves as a potent and physiologically important regulator of mineralization, with systemic and local concentrations determined by several key regulators, including: tissue-nonspecific alkaline phosphatase (ALPL gene; TNAP protein), the progressive ankylosis protein (ANKH; ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1; ENPP1). Results to date have indicated important roles for PPi in cementum formation, and we addressed several gaps in knowledge by employing genetically edited mouse models where PPi metabolism was disrupted and pharmacologically modulating PPi in a PPi-deficient mouse model. We demonstrate that acellular cementum growth is inversely proportional to PPi levels, with reduced cementum in Alpl KO (increased PPi levels) mice and excess cementum in Ank KO mice (decreased PPi levels). Moreover, simultaneous ablation of Alpl and Ank results in reestablishment of functional cementum in dKO mice. Additional reduction of PPi by dual deletion of Ank and Enpp1 does not further increase cementogenesis, and PDL space is maintained in part through bone modeling/remodeling by osteoclasts. Our results provide insights into cementum formation and expand our knowledge of how PPi regulates cementum. We also demonstrate for the first time that pharmacologic manipulation of PPi through an ENPP1-Fc fusion protein can regulate cementum growth, supporting therapeutic interventions targeting PPi metabolism.

Extraskeletal Calcifications in Hutchinson-Gilford Progeria Syndrome.

PURPOSE: Children with Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disease, exhibit extraskeletal calcifications detected by radiographic analysis and on physical examination. The aim of this study was to describe the natural history and pathophysiology of these abnormal calcifications in HGPS, and to determine whether medications and/or supplements tested in clinical trials alter their development. METHODS: Children from two successive clinical trials administering 1) lonafarnib (n = 26) and 2) lonafarnib + pravastatin + zoledronic acid (n = 37) were studied at baseline (pre-therapy), one year on therapy, and at end-of-therapy (3.3-4.3 years after the baseline visit). Calcium supplementation (oral calcium carbonate) was administered during the first year of the second trial and was subsequently discontinued. Information on calcifications was obtained from physical examinations, radiographs, and serum and urinary biochemical measures. The mineral content of two skin-derived calcifications was determined by x-ray diffraction. RESULTS: Extraskeletal calcifications were detected radiographically in 12/39 (31%) patients at baseline. The odds of exhibiting calcifications increased with age (p = 0.045). The odds were unaffected by receipt of lonafarnib, pravastatin, and zoledronate therapies. However, administration of calcium carbonate supplementation, in conjunction with all three therapeutic agents, significantly increased the odds of developing calcifications (p = 0.009), with the odds plateauing after the supplement's discontinuation. Composition analysis of calcinosis cutis showed hydroxyapatite similar to bone. Although serum calcium, phosphorus, and parathyroid hormone (PTH) were within normal limits at baseline and on-therapy, PTH increased significantly after lonafarnib initiation (p < 0.001). Both the urinary calcium/creatinine ratio and tubular reabsorption of phosphate (TRP) were elevated at baseline in 22/39 (56%) and 31/37 (84%) evaluable patients, respectively, with no significant changes while on-therapy. The mean calcium × phosphorus product (Ca × Pi) was within normal limits, but plasma magnesium decreased over both clinical trials. Fibroblast growth factor 23 (FGF23) was lower compared to age-matched controls (p = 0.03). CONCLUSIONS: Extraskeletal calcifications increased with age in children with HGPS and were composed of hydroxyapatite. The urinary calcium/creatinine ratio and TRP were elevated for age while FGF23 was decreased. Magnesium decreased and PTH increased after lonafarnib therapy which may alter the ability to mobilize calcium. These findings demonstrate that children with HGPS with normal renal function and an unremarkable Ca × Pi develop extraskeletal calcifications by an unidentified mechanism that may involve decreased plasma magnesium and FGF23. Calcium carbonate accelerated their development and is, therefore, not recommended for routine supplementation in these children.

Desmoplastic melanoma: a 12-year experience with sentinel lymph node biopsy.

AIMS: Given the paucity of data regarding nodal involvement in desmoplastic melanoma (DM), we decided to review the incidence of nodal metastasis in our patients with DM to better define guidelines regarding the performance of sentinel lymph node biopsy (SLNB) in this specific melanoma subtype. METHODS: Using a prospectively maintained database, we reviewed all patients who underwent treatment for melanoma at the Yale Melanoma Unit in a twelve-year period (1998-2010), during which 3531 cases were treated. We identified 24 patients (0.7%) diagnosed with DM. These patients' records were studied for clinical and histologic parameters and clinical outcomes. RESULTS: Twenty-two patients from the DM group had SLNB, of which four (18%) were diagnosed with micro-metastasis. These four patients were all treated with completion lymphadenectomy and none had additional positive nodes in the remainder of the nodes. Patients were followed after surgery for a median of 25 months (range 2-60 months). Two patients (9%) developed local recurrence, two (9%) in-transit recurrence, and six (27%) showed distant metastases (three patients were pure DM and three patients showed mixed morphology). Patients with mixed DM had a higher rate of nodal metastasis (25%) vs those with pure DM (14%). CONCLUSIONS: Other authors have reported that patients diagnosed with pure DM were less likely to have a positive SLN (0-2%) than those patients with the mixed DM subtype (12-16%). Our findings of higher incidence rates of regional lymph node metastases in both the pure and mixed DM subtypes (14% and 25%) compel us to continue to still recommend that SLNB be considered in patients with both subcategories, pure and mixed DM. LEVEL OF EVIDENCE: Level IV.

Biophysical characterization of gp41 aggregates suggests a model for the molecular mechanism of HIV-associated neurological damage and dementia.

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.

Treatment of autoimmune premature ovarian failure.

There is no known immunosuppressive therapy for autoimmune premature ovarian failure that has been proven safe and effective by prospective randomized placebo-controlled study. Nevertheless, immunosuppression using corticosteroids has been used on an empirical basis for this condition. Here we present two cases of young women with premature ovarian failure who were treated with glucocorticoids in the hopes of restoring fertility. The first case illustrates the potential benefit of such therapy, and the second case illustrates a potential risk. The first patient with histologically proven autoimmune oophoritis was treated with alternate day glucocorticoid treatment. She had return of menstrual bleeding six times and ovulatory progesterone concentrations four times over a 16 week period. The second patient with presumed but unconfirmed autoimmune ovarian failure was referred to us after having been treated with a 9 month course of corticosteroids. During that treatment her menses did not resume. The corticosteroid treatment was complicated by iatrogenic Cushing syndrome and osteonecrosis of the knee. Identifying patients with autoimmune premature ovarian failure presents the opportunity to restore ovarian function by treating these patients with the proper immune modulation therapy. On the other hand, potent immune modulation therapy can have major complications. Corticosteroid therapy for autoimmune premature ovarian failure should be limited to use in placebo-controlled trials designed to evaluate the safety and efficacy of such treatment.

Structure-function relationships in side chain lactam cross-linked peptide models of a conserved N-terminal domain of apolipoprotein E.

Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.

Conformationally specific enhancement of receptor-mediated LDL binding and internalization by peptide models of a conserved anionic N-terminal domain of human apolipoprotein E.

In this paper, we test the hypothesis that peptide models of a highly conserved domain of apolipoprotein E (amino acids 41-60 in human apo E) modulate the binding and internalization of LDL to cell surface receptors in a conformationally specific manner. Three peptides were compared: peptide I containing the natural sequence of amino acids 41-60 of human apo E; peptide III containing side-chain lactam cross-links designed to enhance alpha-helical structure; and peptide II containing cross-links designed to prevent formation of alpha-helices. Peptide III was shown previously to consist of two short alpha-helical domains linked by a turn and to have more alpha-helical content than peptide I, while peptide II was shown to have less helical content than either peptide III or I(Luo et al., 1994). Peptide III induced a 30-fold increase in the specific binding of 125I-LDL to normal human skin fibroblasts and a 60-fold increase in the binding to fibroblasts lacking the LDL-R. This same peptide also restored the binding to normal fibroblasts of 125I-LDL from a patient with familial defective apolipoprotein B, the R3500-->Q mutation. Analysis of binding indicated an increase in the apparent number of binding sites, with little effect on the affinity of 125I-LDL for the cell surface. Heparinase treatment of the cells did not abrogate this effect, suggesting that the increased binding is not mediated by cell surface glycans. LDL internalization but not degradation was also increased by peptide III. Similar but smaller effects were also induced by peptide I. Peptide II was much less active than peptide I or III. Thus, the order of biological activity was the same as the order of alpha-helical content, i.e., peptide III > peptide I > peptide II. These results suggest a hitherto unknown biological function for a highly conserved domain of apolipoprotein E, and this bioactivity was shown by peptide models to be specific to the alpha-helical conformation.

Structural and thermodynamic characterization of a bioactive peptide model of apolipoprotein E: side-chain lactam bridges to constrain the conformation.

Apolipoprotein E plays a critical role in plasma lipoprotein clearance. A peptide model of a highly conserved domain of this protein has been shown to increase low-density lipoprotein binding to fibroblast cell surface receptors. To distinguish between two potential structures--one essentially alpha-helical and nonamphiphilic, the other an amphiphilic pi-helix--synthetic side-chain lactam constraints have been incorporated into model peptides in order to restrict conformational flexibility favoring either the alpha- or pi-helix. Here we provide CD and 1H NMR data suggesting that the more biologically active, putatively alpha-helical peptide indeed contains two alpha-helical domains separated by a central bend. Whereas previous studies (Osapay & Taylor, 1992; Felix et al., 1988) indicated stabilization of alpha-helices by cross-links between the i and i + 4 residues, the current paper demonstrates that cross-links between the i and i + 3 residues also stabilize the helix. Indeed, the stabilization afforded by these cross-links is approximately 1 kcal/mol, similar to that reported for peptides cross-linked between the i and i + 4 residues, and derives exclusively from a loss of entropy of the unfolded state. The presence of the alpha-helical structure appears to correlate well with biological activity. This study provides initial insight into the bioactive structure of this domain of apo E and suggests strategies as to how peptides can be conformationally constrained to enhance their stability and biological function.