Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE^−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Atherosclerotic plaque rupture is the leading cause of myocardial infarction and stroke (1, 2). Studies assessing the correlation between calcium scores and cardiovascular events have demonstrated a predictive power that is superior to and independent from that of lipid scores (3, 4). Additionally, clinical imaging studies have revealed that the risk of plaque rupture is further heightened by the presence of small, “spotty” calcifications, or microcalcifications (5, 6), and cardiovascular risk is inversely correlated with the size of calcific deposits, quantified as a calcium density score (7). Indeed, computational modeling has demonstrated that, while large calcifications can reinforce the fibrous cap (8), microcalcifications (typically 5 to 15 μm in diameter) uniquely mediate an increase in mechanical stress of the relatively soft, collagen-rich fibrous cap (9–12).Histologic studies have revealed the presence of cell-derived vesicles within calcifying atherosclerotic lesions (13–16). The inflammatory environment of the atherosclerotic lesion can induce vascular smooth muscle cells (vSMCs) to take on an osteochondrogenic phenotype and release calcifying extracellular vesicles (EVs) (17–19). Macrophages have also been shown to release procalcifying vesicles (20, 21). Thus, just as bone formation is hypothesized to be an active, cell-driven process (22, 23), mediated by calcifying matrix vesicles, atheroma-associated calcification may similarly be initiated by the production and aggregation of calcifying EVs (11, 20, 24–28).One proposed strategy for halting pathologic calcification has been the use of bisphosphonates (BiPs). BiPs are analogs of pyrophosphate (29), a naturally occurring compound derived in vivo from adenosine triphosphate (ATP) (30). Pyrophosphate binds to calcium phosphate and inhibits calcification via physicochemical mechanisms, namely, by blocking calcium and phosphate ions from forming crystals, preventing crystal aggregation, and preventing mineral transformation from amorphous calcium phosphate to hydroxyapatite (29). BiPs were identified as pyrophosphate analogs that, unlike pyrophosphate itself, resist enzymatic hydrolysis. A second, distinct property of BiPs is the ability to inhibit bone resorption via biological activity directed against osteoclasts following osteoclast endocytosis of the BiP molecule adsorbed to the surface of bone (29, 31). First-generation, or nonnitrogen-containing BiPs, are incorporated into nonhydrolyzable ATP analogs, and induce osteoclast apoptosis by limiting ATP-dependent enzymes. In contrast, nitrogen-containing BiPs inhibit farnesyl pyrophosphate synthetase and thereby induce osteoclast apoptosis (31).In vivo animal investigations have been performed to explore the potential for BiPs to inhibit cardiovascular calcification. Studies of first-generation BiPs revealed that the doses required to inhibit cardiovascular calcification also critically compromised normal bone mineralization (29, 32). However, newer, nitrogen-containing BiPs effectively arrested cardiovascular calcification in animal models at doses that did not compromise bone formation (32). Further, while it has been proposed that BiP treatment modifies cardiovascular calcification via its impact on bone-regulated circulating calcium and phosphate levels, a study in uremic rats demonstrated that BiP treatment inhibited medial aortic calcification with no significant change in plasma calcium and phosphate levels (33). The same study demonstrated that BiP treatment inhibited calcification of explanted rat aortas, indicating that BiPs can act directly on vascular tissue, independent of bone metabolism (33).Retrospective clinical data examining the effect of BiP therapy on cardiovascular calcification has demonstrated conflicting findings and intriguing paradoxes. In women with chronic kidney disease, BiP therapy decreased the mortality rate for patients without a prior history of cardiovascular disease (34), but for those patients with a history of prior cardiovascular events, BiP therapy was associated with an increased mortality rate (35). In another study, BiP therapy correlated with a lower rate of cardiovascular calcification in older patients (>65 y), but a greater rate in younger patients (<65 y) (36). These clinical findings motivated our study, in which we sought to further understand how BiP therapy impacts cardiovascular outcomes. Given that cardiovascular calcification, and especially the presence of microcalcification, is a strong and independent risk factor for adverse cardiac events, and BiPs are prescribed to modulate pathologies of mineralization, we hypothesize that BiPs modulate cardiovascular outcomes by altering the dynamics of cardiovascular calcification.EVs are smaller than the resolution limits of traditional microscopy techniques, hindering studies into the mechanisms of calcification nucleation and growth. We previously developed an in vitro collagen hydrogel platform that allowed the visualization of calcific mineral development mediated by EVs isolated from vSMCs (24). Using superresolution microscopy, confocal, and electron microscopy techniques, we showed that calcification requires the accumulation of EVs that aggregate and merge to build mineral. Collagen serves as a scaffold that promotes associations between EVs that spread into interfibrillar spaces. The resultant mineral that forms within the collagen hydrogel appears spectroscopically similar to microcalcifications in human tissues and allows the study of these structures on the time scale of 1 wk. In this study, we utilized this three-dimensional (3D) acellular platform to examine the direct effect of ibandronate, a nitrogen-containing BiP, on the EV-directed nucleation and growth of microcalcifications, a process that cannot be isolated from cellular and tissue-level mechanisms in a more complex, in vivo system. In parallel, we utilized a mouse model of atherosclerosis to assess the effect of ibandronate therapy on plaque-associated calcification, comparing mineral morphologies between the in vitro and in vivo samples. We hypothesize that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Understanding the EV-specific action of BiPs is imperative both to develop anticalcific therapeutics targeting EV mineralization and to understand one potential mechanism driving the cardiovascular impact of BiPs used in clinical settings.     

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.

The polyprotein Gag is the main structural component of HIV-1, consisting of the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 domains as well as the spacer peptides SP1 and SP2 (1). Gag is produced in infected host cells and trafficked to the plasma membrane, where it assembles into a hexagonal lattice via its CA domain and recruits other viral proteins and the viral RNA genome (1, 2). Assembly of the curved Gag lattice is commensurate with membrane bending at the site of assembly, after which recruitment of Endosomal Sorting Complex Required for Transport III (ESCRT-III) components by the p6 domain of Gag induces membrane scission and release of the immature virus particle (2). The hexagonal Gag lattice accommodates curvature in the growing bud by incorporating vacancy defects (3). The activity of ESCRT-III is timed such that the final immature lattice is incomplete, giving rise to an additional large gap in the lattice, resulting in a truncated spherical shape (4–6).During or after budding, the viral protease is activated and cleaves this immature Gag lattice into its component domains, which leads to structural rearrangement within the virus particle (2). The released CA domains assemble to form a closed, conical capsid around the condensed ribonucleoprotein (RNP) complex of the mature virus (1, 7). Maturation is required for the virion to become infectious (1).Within the immature virus particle, the N-terminal domain of CA (CA_NTD) forms trimeric interactions linking three Gag hexamers while the C-terminal domain of CA (CA_CTD) forms dimeric interactions mediated by helix 9 of CA, linking two Gag hexamers together (8). The CA_CTD additionally forms intrahexamer interactions around the sixfold axis of the hexamer (8, 9). Amphipathic helices formed by the C-terminal residues of CA_CTD and the N-terminal residues of SP1 junction assemble into a six-helix bundle (6HB), thereby imposing hexagonal order on the CA domains, via classical knobs-in-holes packing mediated by exposed hydrophobic side chains, as also seen in coiled coils (9, 10). In combination, these relatively weak interactions give rise to a very dynamic, reversible assembly process that prevents the assembling lattice from becoming trapped in kinetically unfavorable states (11). This robust assembly behavior is consistent with icosahedral viruses (12–15). It is not surprising, therefore, that the energetics of Gag assembly are tightly controlled and highly dependent on scaffolding effects from the viral RNA and the membrane-interacting MA domain of Gag in order to ensure productive viral assembly (11, 16). Analysis of the diffusion pattern of fluorescently labeled Gag supports the notion that Gag is trafficked to the site of assembly as low-order multimers, although it is still unclear whether these are Gag dimers, trimers, or other multimeric forms of Gag (16, 17).The primary assembly unit of the Gag lattice remains largely unknown. We can identify two hypothetical ways in which the lattice could assemble. First, the lattice could grow by addition of Gag hexamers (or sets of six component monomers), such that the CA-SP1 junction is assembled within a hexameric 6HB at all positions in the lattice. In this case, interfaces between hexamers would be unoccupied at the edge of the lattice. From a purely energetic perspective, this appears most reasonable. Second, the lattice could form via addition of Gag dimers or Gag trimers (or equivalently from sets of either two or three component monomers). This would maintain, for example, the dimeric CA-CA interhexamer interactions but leave incomplete hexamers at the lattice edges, including unoccupied hexamer-forming interfaces along the CA-SP1 bundle. It additionally remains unclear whether the unoccupied Gag-Gag interfaces at the lattice edges are simply exposed, or whether they are stabilized by alternative conformations of individual domains or proteins, or by other binding partners. Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus assembly.Viral assembly, budding, and maturation are tightly linked, and disrupting the kinetics of any of these processes can give rise to defects in maturation and formation of noninfectious viral particles (1, 18, 19). The rate-limiting proteolytic cleavage site in the maturation process resides within the CA-SP1 6HB (20). Unfolding of the helical bundle is required to allow proteolytic cleavage to proceed (21–23), but the exact mechanism for protease access to this site is not known. The spatial localization of proteolytic processing within the context of the immature Gag lattice is relevant: Does the protease act on Gag within the lattice, or does it act on the edges of the Gag lattice, causing a cascade of lattice disruption? At the lattice edge, is the substrate for the protease with a 6HB or within an incomplete hexamer? Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus maturation.High-resolution immature Gag structures have previously been determined directly from purified viruses by cryo-electron tomography (cryo-ET) and subtomogram averaging (10). These structures represent an average hexamer within the immature lattice, with a full complement of six Gag hexamer neighbors. Here, we have applied subtomogram classification and averaging approaches to an existing immature virus dataset (10) in order to determine the structures of Gag assemblies at lattice edges. We also applied atomistic molecular dynamics simulations to assess the roles of the different CA-CA interactions in immature lattice stabilization and predict the properties of the structures we observe at lattice edges. Together, our results suggest that the basic unit of immature HIV-1 assembly is a Gag dimer and partial CA-SP1 helical bundles are present at the edges of the assembled lattice and may be substrates for initiation of maturation.     

Unusual case of camptocormia triggered by lumbar-disc herniation

A 21-year-old male patient with low back pain and marked forward bending was presented. The exaggerated lumbar flexion was preventing him to stand in erect posture but disappeared while lying. The symptoms had begun after he had lifted a heavy object. Straight-leg-raising test could not be performed properly because of the exaggerated pain. The light-touch sense was decreased on L5 and S1 dermatomes. There was no loss of muscle strength. The deep-tendon reflexes were normal. Plain graph showed mild narrowing in the L4–5 and L5–S1 intervertebral spaces. Lumbar magnetic resonance imaging revealed disc protrusions in L4–5 and L5–S1 levels. During his stay in the department, the patient was given tizanidine and tramadol, and physical therapy was performed. A paravertebral intramuscular injection with lidocaine was applied. Moreover, the patient was referred to psychiatrist for evaluation regarding his medical history of conversive seizures and possible efforts for secondary gain. No response was obtained from all the treatments. The final diagnosis was camptocormia triggered by lumbar-disc herniation. He was applied supportive psychotherapy, psychoeducation regarding secondary gain, strong suggestions to improve posture, positive reinforcement, and behavioral therapy. His postural abnormality resolved and disappeared completely with mild pain. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

川芎嗪和大黄酸联用对大鼠肝纤维化的抑制作用

研究拟用大黄酸和川芎嗪两种单体联用(简称芎黄联用)，观察二者联用的抗纤维化效应，并探讨其作用机制。     

Ciliary central microtubular orientation is of no clinical significance in bronchiectasis

It has been suggested that patients with bronchiectasis might have increased central microtubular orientation angle (CMOA), which leads to poor coordination of ciliary beating, and consequently impairment of airway defence. We have employed transmission electron microscopy to assess CMOA of ciliated nasal mucosa in a cohort of 133 (81F, 56.8+/-16.1yr) stable bronchiectasis and 59 healthy subjects (30F, 49.3+/-22.1yr). There was no significant difference in CMOA between bronchiectasis (13.2 degree) and control subjects (13.0 degree, P=0.82). There was no significant difference in CMOA among patients according to the etiology of bronchiectasis, presence of nasal symptoms, or sputum status of Pseudomonas aeruginosa infection. Patients with more severe bronchiectasis, i.e. those with FEV(1) <60%, FVC <60%, or more than 4 bronchiectatic lung lobes, had significantly lower CMOA than their counterparts (P<0.05). There was no correlation between CMOA with age, 24h sputum volume, exacerbation frequency, FEV(1), FVC, or the number of bronchiectatic lung lobes (P>0.05). CMOA correlated with ciliary beat frequency (negative), and the percent of cilia showing ultrastructural or microtubular defects (P<0.05). Central microtubular orientation angle does not correlate with clinically important parameters, in contrary to the results reported by previously published smaller scale studies.     

Reading depends on writing, in Chinese

Language development entails four fundamental and interactive abilities: listening, speaking, reading, and writing. Over the past four decades, a large body of evidence has indicated that reading acquisition is strongly associated with a child's listening skills, particularly the child's sensitivity to phonological structures of spoken language. Furthermore, it has been hypothesized that the close relationship between reading and listening is manifested universally across languages and that behavioral remediation using strategies addressing phonological awareness alleviates reading difficulties in dyslexics. The prevailing view of the central role of phonological awareness in reading development is largely based on studies using Western (alphabetic) languages, which are based on phonology. The Chinese language provides a unique medium for testing this notion, because logographic characters in Chinese are based on meaning rather than phonology. Here we show that the ability to read Chinese is strongly related to a child's writing skills and that the relationship between phonological awareness and Chinese reading is much weaker than that in reports regarding alphabetic languages. We propose that the role of logograph writing in reading development is mediated by two possibly interacting mechanisms. The first is orthographic awareness, which facilitates the development of coherent, effective links among visual symbols, phonology, and semantics; the second involves the establishment of motor programs that lead to the formation of long-term motor memories of Chinese characters. These findings yield a unique insight into how cognitive systems responsible for reading development and reading disability interact, and they challenge the prominent phonological awareness view.     

Comparison of the Masaoka-Koga staging and the International Association for the Study of Lung Cancer/the International Thymic Malignancies Interest Group proposal for the TNM staging systems based on the Chinese Alliance for Research in Thymomas retrospective database

Background

To compare the predictive effect of the Masaoka-Koga staging system and the International Association for the Study of Lung Cancer (IASLC)/the International Thymic Malignancies Interest Group (ITMIG) proposal for the new TNM staging on prognosis of thymic malignancies using the Chinese Alliance for Research in Thymomas (ChART) retrospective database.

Methods

From 1992 to 2012, 2,370 patients in ChART database were retrospectively reviewed. Of these, 1,198 patients with complete information on TNM stage, Masaoka-Koga stage, and survival were used for analysis. Cumulative incidence of recurrence (CIR) was assessed in R0 patients. Overall survival (OS) was evaluated both in an R0 resected cohort, as well as in all patients (any R status). CIR and OS were first analyzed according to the Masaoka-Koga staging system. Then, they were compared using the new TNM staging proposal.

Results

Based on Masaoka-Koga staging system, significant difference was detected in CIR among all stages. However, no survival difference was revealed between stage I and II, or between stage II and III. Stage IV carried the highest risk of recurrence and worst survival. According to the new TNM staging proposal, CIR in T1a was significantly lower comparing to all other T categories (P<0.05) and there is a significant difference in OS between T1a and T1b (P=0.004). T4 had the worst OS comparing to all other T categories. CIR and OS were significantly worse in N (+) than in N0 patients. Significant difference in CIR and OS was detected between M0 and M1b, but not between M0 and M1a. OS was almost always statistically different when comparison was made between stages I–IIIa and stages IIIb–IVb. However, no statistical difference could be detected among stages IIIb to IVb.

Conclusions

Compared with Masaoka-Koga staging, the IASLC/ITMIG TNM staging proposal not only describes the extent of tumor invasion but also provides information on lymphatic involvement and tumor dissemination. Further study using prospectively recorded information on the proposed TNM categories would be helpful to better grouping thymic tumors for predicting prognosis and guiding clinical management.     

Angiotensin-I converting enzyme insertion/deletion polymorphism and its association with diabetic nephropathy: a meta-analysis of studies reported between 1994 and 2004 and comprising 14,727 subjects

Aims/hypothesis The ACE insertion/deletion polymorphism has been examined for association with diabetic nephropathy over the past decade with conflicting results. To clarify this situation, we conducted a comprehensive meta-analysis encompassing all relevant studies that were published between 1994 and 2004 and investigated this potential genetic association.Methods A total of 14,727 subjects from 47 studies was included in this meta-analysis. Cases (n=8,663) were type 1 or 2 diabetic subjects with incipient (microalbuminuria) or advanced diabetic nephropathy (proteinuria, chronic renal failure, end-stage renal disease). Control subjects (n=6,064) were predominantly normoalbuminuric.Results No obvious publication bias was detected. Using a minimal-case definition based on incipient diabetic nephropathy, subjects with the II genotype had a 22% lower risk of diabetic nephropathy than carriers of the D allele (pooled odds ratio [OR]=0.78, 95% CI=0.69–0.88). While there was a reduced risk of diabetic nephropathy associated with the II genotype among Caucasians with either type 1 or type 2 diabetes, the association was most marked among type 2 diabetic Asians (Chinese, Japanese, Koreans) (OR=0.65, 95% CI=0. 51–0.83). This OR is significantly different from the OR of 0.90 (95% CI= 0.78–1.04) that was obtained for type 2 diabetic Caucasians (p=0.019). Using a stricter case definition based on advanced diabetic nephropathy, a comparable risk reduction of 24–32% was observed among the three subgroups, although statistical significance was reached only among Asians.Conclusions/interpretation The results of our meta-analysis support a genetic association of the ACE Ins/Del polymorphism with diabetic nephropathy. These findings may have implications for the management of diabetic nephropathy using ACE inhibitors especially among type 2 diabetic Asians.     

红景天与绛香对心肌梗死大鼠血管抑素和内皮抑素表达的影响

目的观察红景天、绛香对缺血心肌中内皮抑素和血管抑素表达的影响,探讨红景天、绛香促血管生成作用的机制.方法雄性Sprague-Dawley(SD)大鼠,按标准方法结扎左冠状动脉前降支,随机分为假手术组和心肌梗死组,心肌梗死组再随机分为对照组、红景天组和绛香组;假手术组和对照组于造模后每天给予生理盐水灌胃;红景天组和绛香组分别于造模后给予红景天、绛香水煎剂灌胃.采用Westernblott法检测心肌组织的血管抑素水平.采用双抗体夹心ELISA法,测定心肌内皮抑素浓度.结果假手术组和对照组比较,绛香组与红景天组缺血心肌内血管数目明显增加(P＜0.05),两组比较无统计学意义(P＞0.05);红景天组和绛香组大鼠梗死边缘区缺血心肌内血管抑素的表达较对照组轻度减少,高于假手术组,红景天组和绛香组之间内皮抑素表达差异不明显.红景天组和绛香组内皮抑素的表达量高于假手术组(P＜0.05),两组比较无统计学意义(P＞0.05).结论红景天、绛香能够明显增加梗死边缘区心肌内血管密度,同时轻度降低血管抑素和内皮抑素的表达,提示红景天和绛香在一定程度上改变了缺血心肌中血管新生的平衡,使血管生长的各因子的平衡向促进血管新生的方向发展.     

双对吻挤压与经典挤压技术治疗冠状动脉分叉病变的前瞻性、随机、多中心研究

目的 比较双对吻挤压(DK crush)和经典挤压技术治疗冠状动脉分叉病变的临床效果.方法 311例真性分叉病变患者随机分入DK crush组(n=155)和经典挤压组(n=156),随访时间8个月.一级及二级终点分别为主要心脏不良事件(MACE,包括心肌梗死、心原性死亡和靶病变血运重建)和血管直径再狭窄及晚期丢失.结果 DK crush组糖尿病患者较多.经典挤压组及DKcrush组最终对吻扩张(FKBI)成功率分别为76%和100%(P＜0.001).DK crush术式的不足包括造影剂用量大(P=0.04)、球囊数量多(P＜0.01)、手术时间长(P＜0.001),但是对吻扩张不满意率显著减少(27.6%比6.3%,P＜0.01).临床随访率为100%,冠状动脉造影随访率为82%.经典挤压组累计再狭窄率为32.3%,而DK crush组为20.3%(P=0.01),经典挤压组分支血管再狭窄率高(24.4%比12.3%,P=0.01),而两组间主干血管再狭窄率差异无统计学意义.经典挤压组术后8个月时的累计MACE发生率为24.4%(FKBI失败组为35.9%,FKBI成功组为19.7%),显著高于DK crush组(11.4%,P=0.02).经典挤压组血栓栓塞率为3.2%(FKBI失败组为5.1%,FKBI成功组为1.7%),而DK crush组为1.3%(P＞0.05).经典挤压组术后8个月时无靶病变血运重建生存率为75.4%(FKBI失败组为71.2%,FKBI成功组为77.6%),而DK crush组为89.5%(P=0.002).结论 DK crush可能是治疗冠状动脉分叉病变的较佳术式.