Cxcl12 Deletion in Mesenchymal Cells Increases Bone Turnover and Attenuates the Loss of Cortical Bone Caused by Estrogen Deficiency in Mice

CXCL12 is abundantly expressed in reticular cells associated with the perivascular niches of the bone marrow (BM) and is indispensable for B lymphopoiesis. Cxcl12 promotes osteoclastogenesis and has been implicated in pathologic bone resorption. We had shown earlier that estrogen receptor α deletion in osteoprogenitors and estrogen deficiency in mice increase Cxcl12 mRNA and protein levels in the BM plasma, respectively. We have now generated female and male mice with conditional deletion of a Cxcl12 allele in Prrx1 targeted cells (Cxcl12^∆Prrx1) and show herein that they have a 90% decrease in B lymphocytes but increased erythrocytes and adipocytes in the marrow. Ovariectomy increased the expression of Cxcl12 and B-cell number in the Cxcl12^f/f control mice, but these effects were abrogated in the Cxcl12^∆Prrx1 mice. Cortical bone mass was not affected in Cxcl12^∆Prrx1 mice. Albeit, the cortical bone loss caused by ovariectomy was greatly attenuated. Most unexpectedly, the rate of bone turnover in sex steroid–sufficient female or male Cxcl12^∆Prrx1 mice was dramatically increased, as evidenced by a more than twofold increase in several osteoblast- and osteoclast-specific mRNAs, as well as increased mineral apposition and bone formation rate and increased osteoclast number in the endosteal surface. The magnitude of the Cxcl12^∆Prrx1-induced changes were much greater than those caused by ovariectomy or orchidectomy in the Cxcl12^f/f mice. These results strengthen the evidence that CXCL12 contributes to the loss of cortical bone mass caused by estrogen deficiency. Moreover, they reveal for the first time that in addition to its effects on hematopoiesis, CXCL12 restrains bone turnover—without changing the balance between resorption and formation—by suppressing osteoblastogenesis and the osteoclastogenesis support provided by cells of the osteoblast lineage. © 2020 American Society for Bone and Mineral Research.     

Letter to the editor concerning “A systematic review and meta-analysis on the management of accidental dural tears in spinal surgery: drowning in information but thirsty for a clear message” by Alshameeri ZAF,et al. [Eur Spine J (2020); DOI 10.1007/s00586-020-06401-y]

European Spine Journal -     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.Discovery and engineering of the microbial opsin genes not only has stimulated basic science investigation into the structure–function relationships of proteins involved in light-triggered ion flow but also has opened up opportunities for biological investigation (reviewed in ref. 1) via the technique of optogenetics, which involves targeting these genes and corresponding optical stimuli to control activity within specified types of cells within intact and functioning biological systems. For example, optogenetics has been used to identify causally the brain cells and projections involved in behaviors relevant to memory formation, affective states, and motor function, among many other discoveries (2–4). For the channelrhodopsins, an important member of this protein family widely used in optogenetics (5, 6), the light-activated cation-conducting channel pore has been the subject of structural investigation, both because of curiosity regarding the physical properties of its ion conduction and because the creation of inhibitory channels had been sought for optogenetic applications. Converging lines of work recently achieved the latter goal; resolving the high-resolution structure of channelrhodopsin (7) allowed a principled structure-guided approach to engineering for chloride selectivity by testing an electrostatic model for pore function (8, 9). Subsequently, by screening the genome of the Guillardia theta microbe, two naturally occurring light-gated chloride-conducting channelrhodopsins (10) were identified.Because optogenetic control of behavior has not yet been demonstrated with chloride channelrhodopsins, and to test further integrative ideas regarding pore function from structural considerations as shown here, we sought to design and test the next generation of enhanced chloride channels (iC++ and SwiChR++). Along the way, we provide the initial test of the hypothesis that light-activated channels will be more efficient tools than pumps for optogenetic neuronal inhibition at the cellular level, demonstrate the initial utility of light-gated chloride channels in controlling behavior in freely moving animals, and reveal key principles regarding the functional selectivity of light-gated ion channel pores.     

Estrogen-eluting, phosphorylcholine-coated stent implantation is associated with reduced neointimal formation but no delay in vascular repair in a porcine coronary model.

Estrogen can inhibit intimal proliferation and accelerate endothelial regeneration after angioplasty. This suggests that estrogen may prevent in-stent restenosis. Unlike other therapies to prevent restenosis, estrogen may also not delay endothelial regrowth, thereby avoiding the risk of late stent thrombosis. The purpose of this work was to determine the effect of a 17beta-estradiol-eluting stent on neointimal formation in a porcine model. Each artery of six pigs was randomized to either a control, low-dose, or high-dose 17beta-estradiol-eluting stent. All animals were sacrificed at 30 days for histopathological analysis. There was a 40% reduction in intimal area in the high-dose stents compared with control stents (2.54 +/- 1.0 vs. 4.13 +/- 1.1 mm(2), for high dose vs. control, respectively; P < 0.05). There was complete endothelial regeneration at 30 days and similar inflammatory response to stenting on histopathology in all the stent groups. This is the first study to show that 17beta-estradiol-eluting stents are associated with reduced neointimal formation without affecting endothelial regeneration in the pig model of in-stent restenosis. Estrogen-coated stents may have a potential benefit in the prevention and treatment of in-stent restenosis.     

Predictors of thrombotic complications after placement of the flexible coil stent

The balloon-expandable, stainless steel, flexible coil stent is a useful device for managing acute or threatened closure after percutaneous transluminal coronary angioplasty.^1–5 Use of the device is associated with thrombosis of the stented vessel in a small but important group of patients.^3,6–10 The clinical, angiographic, and procedural factors associated with stent thrombosis with this device are still unknown. The objective of this study was to define predictors of stent thrombosis occurring within the ftrst month after stenting with this device.     

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA⁺ and VLRC⁺ lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.The only two extant taxa of jawless vertebrates (agnatha), lampreys and hagfishes, occupy a unique position in chordate phylogeny and thus are a focal point for studies in comparative immunology. Although jawless vertebrates were shown to reject skin allografts and to produce serum agglutinins to mammalian red blood cells and bacteria (1, 2), the cellular and molecular bases for these adaptive responses remained elusive until the recent identification of their alternative adaptive immune system (3). In contrast to the antigen receptors of jawed vertebrates, whose structural framework is the Ig-domain, the basic building block of agnathan antigen receptors is the leucine-rich repeat (LRR) (4–6). In analogy to the situation in jawed vertebrates, mature genes of so-called variable lymphocyte receptors (VLRs) are combinatorially assembled from different types of genomic donor LRR cassettes; their sequences are inserted into incomplete germ-line VLR genes (4–6). A gene conversion-like process is postulated to underlie the VLR gene assembly (7, 8), through the activity of orthologs of mammalian activation-induced cytidine deaminase (AID), termed cytidine deaminases 1 and 2 (CDA1 and CDA2) (7, 9). As is the case for T-cell receptors (TCRs) and B-cell receptors (BCRs) of jawed vertebrates, combinatorial VLR assembly generates vast repertoires of diverse anticipatory receptors (4–6).Three VLR genes, VLRA, VLRB and VLRC, have been identified in lampreys and hagfishes (3, 9–12). The three VLR isotypes are differentially expressed by three distinct populations of lymphocytes in lampreys (9, 13). The two types of T-like cells of lamprey, VLRA⁺ and VLRC⁺ lymphocytes, are generated in the thymoid, a lymphoepithelial tissue equivalent to the thymus (13–15); this situation is analogous to the development in the thymus of the two distinct αβ and γδ T-cell lineages in jawed vertebrates. The VLRB⁺ cells appear to be generated in hematopoietic tissues outside of the thymoid (14), much like B cells in jawed vertebrates. These findings suggest that these basic pathways of lymphocyte differentiation already existed in a common ancestor of jawed and jawless vertebrates (4–6, 16).The close developmental relationship of the two principal lineages of T lymphocytes in jawed vertebrates is reflected in the unique genomic organization of the TCR genes that encode the four chains of the two different heterodimeric αβ and γδ TCRs (17). Here, we examine the sequence diversity and genomic organization of VLRA and VLRC receptor genes to gain insight into their functional and evolutionary relationship.