Ablation of Vitamin D Signaling in Cardiomyocytes Leads to Functional Impairment and Stimulation of Pro-Inflammatory and Pro-Fibrotic Gene Regulatory Networks in a Left Ventricular Hypertrophy Model in Mice.

The association between vitamin D deficiency and cardiovascular disease remains a controversial issue. This study aimed to further elucidate the role of vitamin D signaling in the development of left ventricular (LV) hypertrophy and dysfunction. To ablate the vitamin D receptor (VDR) specifically in cardiomyocytes, VDRfl/fl mice were crossed with Mlcv2-Cre mice. To induce LV hypertrophy experimentally by increasing cardiac afterload, transverse aortic constriction (TAC) was employed. Sham or TAC surgery was performed in 4-month-old, male, wild-type, VDRfl/fl, Mlcv2-Cre, and cardiomyocyte-specific VDR knockout (VDRCM-KO) mice. As expected, TAC induced profound LV hypertrophy and dysfunction, evidenced by echocardiography, aortic and cardiac catheterization, cardiac histology, and LV expression profiling 4 weeks post-surgery. Sham-operated mice showed no differences between genotypes. However, TAC VDRCM-KO mice, while having comparable cardiomyocyte size and LV fibrosis to TAC VDRfl/fl controls, exhibited reduced fractional shortening and ejection fraction as measured by echocardiography. Spatial transcriptomics of heart cryosections revealed more pronounced pro-inflammatory and pro-fibrotic gene regulatory networks in the stressed cardiac tissue niches of TAC VDRCM-KO compared to VDRfl/fl mice. Hence, our study supports the notion that vitamin D signaling in cardiomyocytes plays a protective role in the stressed heart.

Celebrating 50-years: the history and future of the International Society of Bone Morphometry.

The International Society of Bone Morphometry (ISBM) is dedicated to advancing research, education, and clinical practice for osteoporosis and other bone disorders by developing and improving tools for the quantitative imaging and analysis of bone. Its initial core mission was to promote the proper use of morphometric techniques in bone research and to educate and train clinicians and basic scientists in bone morphometry. This article chronicles the evolution of the ISBM and the history and development of bone morphometric techniques for the past 50-years, starting with workshops on bone morphometry in 1973, to the formal incorporation of the ISBM in 1996, to today. We also provide a framework and vision for the coming decades. This effort was led by ISBM presidents Dr Erica L. Scheller (2022-2024) and Dr Thomas J. Wronski (2009-2012) in collaboration with all other living ISBM presidents. Though the underlying techniques and questions have changed over time, the need for standardization of established tools and discovery of novel approaches for bone morphometry remains a constant. The ISBM fulfills this need by providing a forum for the exchange of ideas, with a philosophy that encourages the open discussion of pitfalls and challenges among clinicians, scientists, and industry partners. This facilitates the rapid development and adaptation of tools to meet emerging demands within the field of bone health at a high level.

Interaction of Vitamin D with Peptide Hormones with Emphasis on Parathyroid Hormone, FGF23, and the Renin-Angiotensin-Aldosterone System.

The seminal discoveries that parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) are major endocrine regulators of vitamin D metabolism led to a significant improvement in our understanding of the pivotal roles of peptide hormones and small proteohormones in the crosstalk between different organs, regulating vitamin D metabolism. The interaction of vitamin D, FGF23 and PTH in the kidney is essential for maintaining mineral homeostasis. The proteohormone FGF23 is mainly secreted from osteoblasts and osteoclasts in the bone. FGF23 acts on proximal renal tubules to decrease production of the active form of vitamin D (1,25(OH)2D) by downregulating transcription of 1α-hydroxylase (CYP27B1), and by activating transcription of the key enzyme responsible for vitamin D degradation, 24-hydroxylase (CYP24A1). Conversely, the peptide hormone PTH stimulates 1,25(OH)2D renal production by upregulating the expression of 1α-hydroxylase and downregulating that of 24-hydroxylase. The circulating concentration of 1,25(OH)2D is a positive regulator of FGF23 secretion in the bone, and a negative regulator of PTH secretion from the parathyroid gland, forming feedback loops between kidney and bone, and between kidney and parathyroid gland, respectively. In recent years, it has become clear that vitamin D signaling has important functions beyond mineral metabolism. Observation of seasonal variations in blood pressure and the subsequent identification of vitamin D receptor (VDR) and 1α-hydroxylase in non-renal tissues such as cardiomyocytes, endothelial and smooth muscle cells, suggested that vitamin D may play a role in maintaining cardiovascular health. Indeed, observational studies in humans have found an association between vitamin D deficiency and hypertension, left ventricular hypertrophy and heart failure, and experimental studies provided strong evidence for a role of vitamin D signaling in the regulation of cardiovascular function. One of the proposed mechanisms of action of vitamin D is that it functions as a negative regulator of the renin-angiotensin-aldosterone system (RAAS). This finding established a novel link between vitamin D and RAAS that was unexplored until then. During recent years, major progress has been made towards a more complete understanding of the mechanisms by which FGF23, PTH, and RAAS regulate vitamin D metabolism, especially at the genomic level. However, there are still major gaps in our knowledge that need to be filled by future research. The purpose of this review is to highlight our current understanding of the molecular mechanisms underlying the interaction between vitamin D, FGF23, PTH, and RAAS, and to discuss the role of these mechanisms in physiology and pathophysiology.

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

Long-Term Excessive Dietary Phosphate Intake Increases Arterial Blood Pressure, Activates the Renin-Angiotensin-Aldosterone System, and Stimulates Sympathetic Tone in Mice.

Increased dietary phosphate intake has been associated with severity of coronary artery disease, increased carotid intima-media thickness, left ventricular hypertrophy (LVH), and increased cardiovascular mortality and morbidity in individuals with normal renal function as well as in patients suffering from chronic kidney disease. However, the underlying mechanisms are still unclear. To further elucidate the cardiovascular sequelae of long-term elevated phosphate intake, we maintained male C57BL/6 mice on a calcium, phosphate, and lactose-enriched diet (CPD, 2% Ca, 1.25% P, 20% lactose) after weaning them for 14 months and compared them with age-matched male mice fed a normal mouse diet (ND, 1.0% Ca, 0.7% P). Notably, the CPD has a balanced calcium/phosphate ratio, allowing the effects of elevated dietary phosphate intake largely independent of changes in parathyroid hormone (PTH) to be investigated. In agreement with the rationale of this experiment, mice maintained on CPD for 14 months were characterized by unchanged serum PTH but showed elevated concentrations of circulating intact fibroblast growth factor-23 (FGF23) compared with mice on ND. Cardiovascular phenotyping did not provide evidence for LVH, as evidenced by unchanged LV chamber size, normal cardiomyocyte area, lack of fibrosis, and unchanged molecular markers of hypertrophy (Bnp) between the two groups. However, intra-arterial catheterization revealed increases in systolic pressure, mean arterial pressure, and pulse pressure in mice fed the CPD. Interestingly, chronically elevated dietary phosphate intake stimulated the renin-angiotensin-aldosterone system (RAAS) as evidenced by increased urinary aldosterone in animals fed the CPD, relative to the ND controls. Furthermore, the catecholamines epinephrine, norepinephrine, and dopamine as well as the catecholamine metabolites metanephrine. normetanephrine and methoxytyramine as measured by mass spectrometry were elevated in the urine of mice on CPD, relative to mice on the ND. These changes were partially reversed by switching 14-month-old mice on CPD back to ND for 2 weeks. In conclusion, our data suggest that excess dietary phosphate induces a rise in blood pressure independent of secondary hyperparathyroidism, and that this effect may be mediated through activation of the RAAS and stimulation of the sympathetic tone.

Activation of RAAS Signaling Contributes to Hypertension in Aged Hyp Mice.

High circulating levels of fibroblast growth factor-23 (FGF23) are associated with left ventricular hypertrophy as well as increased morbidity and mortality in patients suffering from chronic kidney disease. However, the mechanisms underlying this association are controversial. Here, we aimed to further characterize the cardiovascular sequelae of long term endogenous FGF23 hypersecretion using 14-month-old male Hyp mice as a model of FGF23 excess. Hyp mice were characterized by a ~10-fold increase in circulating intact FGF23, hypophosphatemia, increased serum aldosterone, but normal kidney function, relative to wildtype (WT) controls. Cardiovascular phenotyping did not reveal any evidence of left ventricular hypertrophy or functional impairment in 14-month-old Hyp mice. Fractional shortening, ejection fraction, molecular markers of hypertrophy (Anp, Bnp), and intracardiac markers of contractility and diastolic function were all unchanged in these animals. However, intraarterial catheterization revealed an increase in systolic, diastolic, and mean arterial pressure of ~12 mm Hg in aged Hyp mice relative to WT controls. Hypertension in Hyp mice was associated with increased peripheral vascular resistance. To test the hypothesis that a stimulation of the renin-angiotensin-aldosterone system (RAAS) contributes to hypertension in aged Hyp mice, we administered the angiotensin receptor blocker losartan (30 mg/kg twice daily) or the mineralocorticoid receptor antagonist canrenone (30 mg/kg once daily) to aged Hyp and WT mice over 5 days. Both drugs had minor effects on blood pressure in WT mice, but reduced blood pressure and peripheral vascular resistance in Hyp mice, suggesting that a stimulation of the RAAS contributes to hypertension in aged Hyp mice.

FGF23 and Vitamin D Metabolism.

Apart from its phosphaturic action, the bone-derived hormone fibroblast growth factor-23 (FGF23) is also an essential regulator of vitamin D metabolism. The main target organ of FGF23 is the kidney, where FGF23 suppresses transcription of the key enzyme in vitamin D hormone (1,25(OH)2D) activation, 1α-hydroxylase, and activates transcription of the key enzyme responsible for vitamin D degradation, 24-hydroxylase, in proximal renal tubules. The circulating concentration of 1,25(OH)2D is a positive regulator of FGF23 secretion in bone, forming a feedback loop between kidney and bone. The importance of FGF23 as regulator of vitamin D metabolism is underscored by the fact that in the absence of FGF23 signaling, the tight control of renal 1α-hydroxylase fails, resulting in overproduction of 1,25(OH)2D in mice and men. During recent years, big strides have been made toward a more complete understanding of the mechanisms underlying the FGF23-mediated regulation of vitamin D metabolism, especially at the genomic level. However, there are still major gaps in our knowledge that need to be filled by future research. Importantly, the intracellular signaling cascades downstream of FGF receptors regulating transcription of 1α-hydroxylase and 24-hydroxylase in proximal renal tubules still remain unresolved. The purpose of this review is to highlight our current understanding of the molecular mechanisms underlying the regulation of vitamin D metabolism by FGF23, and to discuss the role of these mechanisms in physiology and pathophysiology. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Aldosterone Is Positively Associated With Circulating FGF23 Levels in Chronic Kidney Disease Across Four Species, and May Drive FGF23 Secretion Directly.

BACKGROUND: Chronic kidney disease (CKD) is accompanied by increases in circulating fibroblast growth factor 23 (FGF23) and aldosterone levels. Here, we tested the hypothesis that aldosterone may be one of the driving forces behind increased FGF23 secretion in CKD. METHODS: Using data from a prospective study in humans, a retrospective study in dogs and cats, and an experimental study in 5/6-nephrectomized mice, we analyzed the relationship between circulating FGF23 and serum aldosterone levels in CKD across four species. To assess the effects of acute inhibition of aldosterone signaling on circulating FGF23, we acutely treated mice with established CKD with the mineralocorticoid receptor blocker canrenone (50 mg/kg iv/sc), and measured intact FGF23 before and 24 h as well as 72 h after start of administration of the drug. RESULTS: We found a tight positive association between circulating intact FGF23 and serum aldosterone in human, canine, and feline CKD patients, as well as in experimental murine CKD (humans: r S = 0.57, p = 0.0368; dogs: r S = 0.66, p = 0.0019; cats: r S = 0.75, p = 0.0003; mice: r S = 0.49, p = 0.0004). Injection of canrenone in mice with established CKD did not lead to changes in FGF23 levels within 24 h, but reduced FGF23 in all mice at 72 h. CONCLUSION: Aldosterone may drive enhanced FGF23 secretion in CKD, possibly explaining the tight positive association between circulating intact FGF23 and aldosterone in human, canine, and feline CKD patients as well as in experimental CKD models.

The bone is the major source of high circulating intact fibroblast growth factor-23 in acute murine polymicrobial sepsis induced by cecum ligation puncture.

Fibroblast growth factor-23 (FGF23), a bone-produced hormone, plays a critical role in mineral homeostasis. Human diseases associated with excessive intact circulating FGF23 (iFGF23) result in hypophosphatemia and low vitamin D hormone in patients with normal kidney function. In addition, there is accumulating evidence linking FGF23 with inflammation. Based on these studies and the frequent observation of hypophosphatemia among septic patients, we sought to elucidate further the relationship between FGF23 and mineral homeostasis in a clinically relevant murine polymicrobial sepsis model. Medium-severity sepsis was induced by cecum ligation puncture (CLP) in adult CD-1 mice of both sexes. Healthy CD-1 mice (without CLP) were used as controls. Forty-eight hours post-CLP, spontaneous urine was collected, and serum, organs and bones were sampled at necropsy. Serum iFGF23 increased ~20-fold in CLP compared to control mice. FGF23 protein concentration was increased in the bones, but not in spleen or liver of CLP mice. Despite the ~20-fold iFGF23 increase, we did not observe any significant changes in mineral homeostasis or parathyroid hormone levels in the blood of CLP animals. Urinary excretion of phosphate, calcium, and sodium remained unchanged in male CLP mice, whereas female CLP mice exhibited lower urinary calcium excretion, relative to healthy controls. In line with renal FGF23 resistance, expression of phosphate-, calcium- and sodium-transporting proteins did not show consistent changes in the kidneys of male and female CLP mice. Renal expression of the co-receptor αKlotho was downregulated in female, but not in male CLP mice. In conclusion, our data demonstrate that the dramatic, sex-independent rise in serum iFGF23 post-CLP was mainly caused by an upregulation of FGF23 secretion in the bone. Surprisingly, the upsurge in circulating iFGF23 did not alter humoral mineral homeostasis in the acutely septic mice. Hence, the biological function of elevated FGF23 in sepsis remains unclear and warrants further studies.

Randomized Trial of Etelcalcetide for Cardiac Hypertrophy in Hemodialysis.

[Figure: see text].

Thioredoxin 1 is upregulated in the bone and bone marrow following experimental myocardial infarction: evidence for a remote organ response.

Ischemia and reperfusion events, such as myocardial infarction (MI), are reported to induce remote organ damage severely compromising patient outcomes. Tissue survival and functional restoration relies on the activation of endogenous redox regulatory systems such as the oxidoreductases of the thioredoxin (Trx) family. Trxs and peroxiredoxins (Prxs) are essential for the redox regulation of protein thiol groups and for the reduction of hydrogen peroxide, respectively. Here, we determined whether experimental MI induces changes in Trxs and Prxs in the heart as well as in secondary organs. Levels and localization of Trx1, TrxR1, Trx2, Prx1, and Prx2 were analyzed in the femur, vertebrae, and kidneys of rats following MI or sham surgery. Trx1 levels were significantly increased in the heart (P = 0.0017) and femur (P < 0.0001) of MI animals. In the femur and lumbar vertebrae, Trx1 upregulation was detected in bone-lining cells, osteoblasts, megakaryocytes, and other hematopoietic cells. Serum levels of Trx1 increased significantly 2 days after MI compared to sham animals (P = 0.0085). Differential regulation of Trx1 in the bone was also detected by immunohistochemistry 1 month after MI. N-Acetyl-cysteine treatment over a period of 1 month induced a significant reduction of Trx1 levels in the bone of MI rats compared to sham and to MI vehicle. This study provides first evidence that MI induces remote organ upregulation of the redox protein Trx1 in the bone, as a response to ischemia-reperfusion injury in the heart.

The Role of Natriuretic Peptides in the Regulation of Cardiac Tolerance to Ischemia/Reperfusion and Postinfarction Heart Remodeling.

In the past 10 years, mortality from acute myocardial infarction has not decreased despite the widespread introduction of percutaneous coronary intervention. The reason for this situation is the absence in clinical practice of drugs capable of preventing reperfusion injury of the heart with high efficiency. In this regard, noteworthy natriuretic peptides (NPs) which have the infarct-limiting effect, prevent reperfusion cardiac injury, prevent adverse post-infarction remodeling of the heart. Atrial natriuretic peptide does not have the infarct-reducing effect in rats with alloxan-induced diabetes mellitus. NPs have the anti-apoptotic and anti-inflammatory effects. There is indirect evidence that NPs inhibit pyroptosis and autophagy. Published data indicate that NPs inhibit reactive oxygen species production in cardiomyocytes, aorta, heart, kidney and the endothelial cells. NPs can suppress aldosterone, angiotensin II, endothelin-1 synthesize and secretion. NPs inhibit the effects aldosterone, angiotensin II on the post-receptor level through intracellular signaling events. NPs activate guanylyl cyclase, protein kinase G and protein kinase A, and reduce phosphodiesterase 3 activity. NO-synthase and soluble guanylyl cyclase are involved in the cardioprotective effect of NPs. The cardioprotective effect of natriuretic peptides is mediated via activation of kinases (AMPK, PKC, PI3 K, ERK1/2, p70s6 k, Akt) and inhibition of glycogen synthase kinase 3ß. The cardioprotective effect of NPs is mediated via sarcolemmal KATP channel and mitochondrial KATP channel opening. The cardioprotective effect of brain natriuretic peptide is mediated via MPT pore closing. The anti-fibrotic effect of NPs may be mediated through inhibition TGF-ß1 expression. Natriuretic peptides can inhibit NF-κB activity and activate GATA. Hemeoxygenase-1 and peroxisome proliferator-activated receptor γ may be involved in the infarct-reducing effect of NPs. NPs exhibit the infarct-limiting effect in patients with acute myocardial infarction. NPs prevent post-infarction remodeling of the heart. To finally resolve the question of the feasibility of using NPs in AMI, a multicenter, randomized, blind, placebo-controlled study is needed to assess the effect of NPs on the mortality of patients after AMI.

No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

Osteocytic osteolysis/perilacunar remodeling is thought to contribute to the maintenance of mineral homeostasis. Here, we utilized a reversible, adult-onset model of secondary hyperparathyroidism to study femoral bone mineralization density distribution (BMDD) and osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging. Male mice with a non-functioning vitamin D receptor (VDRΔ/Δ) or wild-type mice were exposed to a rescue diet (RD) (baseline) and subsequently to a low calcium challenge diet (CD). Thereafter, VDRΔ/Δ mice received either the CD, a normal diet (ND), or the RD. At baseline, BMDD and OLS characteristics were similar in VDRΔ/Δ and wild-type mice. The CD induced large cortical pores, osteomalacia, and a reduced epiphyseal average degree of mineralization in the VDRΔ/Δ mice relative to the baseline (-9.5%, p < 0.05 after two months and -10.3%, p < 0.01 after five months of the CD). Switching VDRΔ/Δ mice on the CD back to the RD fully restored BMDD to baseline values. However, OLS remained unchanged in all groups of mice, independent of diet. We conclude that adult VDRΔ/Δ animals on an RD lack any skeletal abnormalities, suggesting that VDR signaling is dispensable for normal bone mineralization as long as mineral homeostasis is normal. Our findings also indicate that VDRΔ/Δ mice attempt to correct a calcium challenge by enhanced osteoclastic resorption rather than by osteocytic osteolysis.

Vitamin D and Cardiovascular Disease, with Emphasis on Hypertension, Atherosclerosis, and Heart Failure.

Vitamin D deficiency is the most common nutritional deficiency, affecting almost one billion people worldwide. Vitamin D is mostly known for its role in intestinal calcium absorption and bone mineralization. However, the observation of seasonal changes in blood pressure and the subsequent identification of vitamin D receptor (VDR) and 1α-hydroxylase in cardiomyocytes, as well as endothelial and vascular smooth muscle cells, implicated a role of vitamin D in the cardiovascular system. Animal studies provided compelling evidence that vitamin D signaling is essential for cardiovascular integrity, especially for the regulation of vascular tone and as an antifibrotic and antihypertrophic signaling pathway in the heart. In addition, observational studies reported an association between vitamin D deficiency and risk of hypertension, atherosclerosis, and heart failure. However, recent clinical intervention studies failed to prove the causal relationship between vitamin D supplementation and beneficial effects on cardiovascular health. In this review, we aim to highlight our current understanding of the role of vitamin D in the cardiovascular system and to find potential explanations for the large discrepancies between the outcome of experimental studies and clinical intervention trials.

Ablation of Vitamin D Signaling Compromises Cerebrovascular Adaptation to Carotid Artery Occlusion in Mice.

Vitamin D insufficiency has been associated with increased incidence and severity of cerebrovascular disorders. We analyzed the impact of impaired vitamin D signaling on the anatomical and functional aspects of cerebrovascular adaptation to unilateral carotid artery occlusion (CAO), a common consequence of atherosclerosis and cause of ischemic stroke. Cerebrocortical blood flow (CoBF) showed a significantly increased drop and delayed recovery after CAO in mice carrying a functionally inactive vitamin D receptor (VDR) with the most sustained perfusion deficit in the temporal cortex. To identify the cause(s) for this altered adaptation, the extent of compensatory blood flow increase in the contralateral carotid artery and the morphology of pial collaterals between the anterior and middle cerebral arteries were determined. Whereas VDR deficiency had no significant influence on the contralateral carotid arterial blood flow increase, it was associated with decreased number and increased tortuosity of pial anastomoses resulting in unfavorable changes of the intracranial collateral circulation. These results indicate that VDR deficiency compromises the cerebrovascular adaptation to CAO with the most sustained consequences in the temporal cortex. The dysregulation can be attributed to the altered development and function of pial collateral circulation whereas extracranial vessels may not be impaired.

Age-related sex differences in the expression of important disease-linked mitochondrial proteins in mice.

The prevalence and progression of many illnesses, such as neurodegenerative and cardiovascular diseases, obesity, and cancer, vary between women and men, often in an age-dependent manner. A joint hallmark of these diseases is some type of mitochondrial dysfunction. While several mitochondrial proteins are known to be regulated by sex hormones, the levels of those proteins have not been systematically analyzed with regard to sex and age, and studies that consider sex and/or age differences in the protein expression are very rare. In this study, we compared the expression patterns of physiologically important mitochondrial proteins in female and male C57BL/6N mice of age cohorts frequently used in experiments. We found that sex-related differences in the expression of uncoupling proteins 1 and 3 (UCP1 and UCP3) occur in an age-dependent manner. The sex-specific expression of UCP1 and UCP3 in brown adipose tissue (BAT) was inversely correlated with differences in body weight. Expression of UCP4 in the brain, Complex I in the spleen, and Complex II in the brain and BAT was least affected by the sex of the mouse. We further demonstrated that there are serious limitations in using VDAC1 and actin as markers in western blot analyses, due to their sex- and age-specific fluctuations. Our results confirm that sex and age are important parameters and should be taken into account by researchers who examine the mechanistic aspects of diseases. HIGHLIGHTS: I.The levels of UCP1 and UCP3 protein expression differ between females and males in an age-dependent manner.II.Pre-pubertal expression of almost all proteins tested in this study does not depend on the sex of the mouse.III.Expression of VDAC1 and actin, which are often used as loading control proteins in western blot analysis, is tissue-specifically influenced by sex and age.

Biomechanical and Bone Material Properties of Schnurri-3 Null Mice.

Schnurri-3 (Shn3) is an essential regulator of postnatal skeletal remodeling. Shn3-deficient mice (Shn3-/-) have high bone mass; however, their bone mechanical and material properties have not been investigated to date. We performed three-point bending of femora, compression tests of L3 vertebrae. We also measured intrinsic material properties, including bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics by quantitative backscatter electron imaging, as well as collagen cross-linking by Fourier transform infrared microspectroscopy of femora from Shn3-/- and WT mice at different ages (6 weeks, 4 months, and 18 months). Moreover, computer modeling was performed for the interpretation of the BMDD outcomes. Femora and L3 vertebrae from Shn3-/- aged 6 weeks revealed increased ultimate force (2.2- and 3.2-fold, p < .01, respectively). Mineralized bone volume at the distal femoral metaphysis was about twofold (at 6 weeks) to eightfold (at 4 and 18 months of age) in Shn3-/- (p < .001). Compared with WT, the average degree of trabecular bone mineralization was similar at 6 weeks, but increased at 4 and 18 months of age (+12.6% and +7.7%, p < .01, respectively) in Shn3-/-. The analysis of OLS characteristics revealed a higher OLS area for Shn3-/- versus WT at all ages (+16%, +23%, +21%, respectively, p < .01). The collagen cross-link ratio was similar between groups. We conclude that femora and vertebrae from Shn3-/- had higher ultimate force in mechanical testing. Computer modeling demonstrated that in cases of highly increased bone volume, the average degree of bone matrix mineralization can be higher than in WT bone, which was actually measured in the older Shn3-/- groups. The area of 2D osteocyte lacunae sections was also increased in Shn3-deficiency, which could only partly be explained by larger remnant areas of primary cortical bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Intra-articularly injected mesenchymal stem cells promote cartilage regeneration, but do not permanently engraft in distant organs.

Intra-articular (IA) injection of mesenchymal stem cells (MSCs) promotes articular cartilage repair. However, cell fate and action after transplantation remain unclear. This study aimed at evaluating the biodistribution and efficacy of MSCs after IA injection. We used an immunocompetent, dual transgenic rat model, which is based on donor rats ubiquitously expressing heat stable human placental alkaline phosphatase (ALPP), and recipient rats expressing a heat sensitive ALPP form. A focal cartilage defect was created in the patellofemoral groove of recipient rats. Bone marrow-derived MSCs isolated from donor rats were injected into the synovial cavity of recipients, and cell tracking was performed in distant organs and knees over 6 months post-injection. A few donor MSCs were observed in the lung of one of the recipients, 1 day post-injection. We failed to detect donor MSCs in any of the studied tissues at all later time points. IA-injected MSCs remained in the synovial cavity, engrafted within the cartilage lesion, and were detectable up to 1 month post-injection. Although the number of MSCs decreased over time, MSCs injection promoted cartilage regeneration as evidenced by histology and immunofluorescent collagen staining. Our study supports the safety and efficacy of using MSCs for cartilage repair via IA delivery.