Osteoblasts are derived from bone marrow mesenchymal stem cells. Hence, some studies sought to establish whether mesenchymal stem cells and their differentiation towards osteoblasts are impaired by HIV infection (Fig. 2). Wang et al.  showed that bone marrow mesenchymal stem cells could be infected to a low extent by X4 tropic HIV strains leading to persistent harbouring of the virus inside these cells with subsequent inhibition of proliferation and survival. Several differentiation pathways from mesenchymal cells are also impaired by Tat through the upregulation of TNF-α and IL-1β expression.
More recently, the interaction between specific HIV proteins and mesenchymal cells differentiating towards osteoblasts was analysed. In particular, p55gag and gp120 viral proteins elicited a derangement of specific transcription factors involved in the differentiation and activity of osteoblasts. HIV gp120 (Fig. 2) is also able to trigger the activation of peroxisome proliferator-activated receptor gamma (PPARγ) determining an MSC differentiation switch from osteoblasts to adipocytes [55,59].
M-CSF is a haematopoietic growth factor controlling the survival, proliferation and differentiation of the monocyte-macrophage lineage and it is closely involved in the early phases of osteoclast differentiation. The pivotal involvement of M-CSF and its receptor in osteoclast differentiation was also confirmed by osteopetrosis and bone alterations in mice mutated in the CSF-1 or c-fms gene [66,67]. HIV infection of macrophages induces a significant increase in M-CSF production and secretion , which in turn promotes further HIV infection of macrophages through the increase in CD4/CCR5 receptors and virus gene expression [69–72]. M-CSF elicits osteoclast differentiation also enhancing the RANKL effect (Fig. 3). In addition, Yamada et al.  showed that bone marrow macrophages (BMMs) cultured without M-CSF produce a large amount of OPG compared with cells cultured with M-CSF. This finding suggests that M-CSF downregulates OPG production in BMMs. As OPG, a TNF receptor family secreting glycoprotein, inhibits osteoclast differentiation by acting as a decoy RANKL receptor, the increasing level of M-CSF during HIV infection impairs the balance between RANKL/RANK and OPG, increasing osteoclasts (Table 2).
In 1995, the introduction of HAART in the treatment of HIV infection led to a dramatic and sustained decrease in HIV-related morbidity and mortality . HAART typically combines nucleoside analogue reverse transcriptase inhibitors (NRTIs) with either HIV protease inhibitors or nonnucleoside reverse transcriptase inhibitors (NNRTIs). Despite controversial results regarding antiretroviral molecules and bone loss, several groups investigated the possible bone damage mechanisms of specific antiretroviral classes.
The nucleoside analogues (NRTIs) are antiretroviral molecules whose chemical structure is a modified nucleoside. These compounds suppress the replication of retroviruses by interfering with the reverse transcriptase enzyme activity causing premature termination of the proviral HIV DNA chain. Abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine and zidovudine are currently used in HAART.
Despite the major positive impact of these molecules in HIV therapy, clinical observations disclosed severe side effects such as mitochondrial toxicity, hyperlactataemia and lactic acidosis. To varying degrees, NRTIs inhibit the DNA polymerase-γ , the enzyme involved in the replication of mitochondrial DNA, leading to mitochondrial damage and dysfunction . In-vitro studies disclosed some differences in the induction of specific NRTI-related mitochondrial DNA depletion. The so-called ‘d-drugs’ ddC (zalcitabine), ddI (didanosine), and d4T (stavudine) are relatively stronger inhibitors of polymerase-γ than other nucleoside analogues, called ‘non-D drugs’ [77,78]. In the presence of mitochondrial dysfunction or depletion, the metabolism of pyruvate is shifted toward the production of lactate with a decrease in energy production. Hyperlactataemia does not inevitably lead to lactic acidosis even though this condition is more prevalent in women, obese individuals, individuals with hepatitis C virus (HCV) coinfection and patients receiving stavudine plus didanosine [79,80]. Several studies based on NRTIs-treated patients demonstrated that hyperlactataemia is a relatively common event occurring in 15–20% of individuals a year, whereas lactic acidosis is encountered in less than 0.4% of patients [79,80].
Although it is commonly classified with NRTIs, tenofovir disoproxil fumarate (TDF) is a nucleotide analogue with anti-HIV activity. In-vitro studies demonstrated that the potential of TDF to cause mitochondrial toxicity is very low compared with other NRTIs [82,83]. Preclinical animal studies have shown that renal excretion is the primary route of TDF elimination by a combination of tubular secretion and glomerular filtration and some evidence of mild nephrotoxicity was noted in different animal species . This renal toxicity is dose-dependent , related to tubular dysfunction due to proximal tubular epithelial cell damage, and correlates with an impaired glomerular filtration rate. The tubular dysfunction may elicit the appearance of hypophosphataemia, whereas the reduced glomerular filtration is associated with a parallel decrease in the function of the alpha-1-hydroxylase, an enzyme involved in vitamin D metabolism . Some papers and case reports have described a nephrotoxicity with hypophosphataemia (Table 3) in HIV-infected patients receiving TDF [87–90] and, in rare cases, the presence of Fanconi syndrome [91,92]. Fanconi syndrome was mainly observed in patients treated with salvage therapy containing ritonavir [93–95] or lopinavir/ritonavir . The impaired phosphorus balance and vitamin D metabolism related to renal toxicity may determine an osteomalacic pattern in HIV patients: some studies found an association between use of TDF and bone damage [97,98] and a higher incidence of foot fracture was also found in TDF-treated patients compared with TDF-untreated individuals [99,100].
The association between TDF and nephrotoxicity was not confirmed by other studies. A cohort study published in 2006  showed a low-grade hypophosphataemia in TDF-treated patients with normal baseline renal function, but it was not statistically significant with respect to TDF-untreated patients. The GS 902  and GS 907 studies , performed on a large number of patients with no history of renal disease, found the same incidences of elevated serum creatinine and hypophosphataemia in the TDF arm and placebo arm after 24 weeks of treatment. The larger GS 903 study, a randomized, double-blind, parallel, placebo-controlled trial over 144 weeks, compared a treatment regimen of TDF, lamivudine and efavirenz with a treatment regimen of stavudine, lamivudine and EFV in antiretroviral-naive patients [104,105], confirming the GS 902 and GS 907 results.
These controversial results may be related to cohort selection, as the patients in the GS studies did not show low GFR at baseline. It is conceivable that the continuous therapeutic regimen, specific HAART pharmacological association and basal functional renal conditions of patients affected the interaction between TDF and bone.
Protease inhibitors impair HIV replication by preventing the viral protease enzymatic action, a pivotal step in the final stages of the viral replication cycle. The viral progeny obtained in the presence of protease inhibitor cannot infect the target cells. Amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir are the protease inhibitors used for antiretroviral therapy.
The protease inhibitors/bone interaction has been studied in in-vitro bone cell cultures (Table 3). The effects of protease inhibitors on osteoclasts were studied measuring osteoclast activity in rat neonatal calvaria . Nelfinavir, indinavir, saquinavir and ritonavir treatments showed proosteoclast activity whereas lopinavir and amprenavir did not. Further studies demonstrated that saquinavir and ritonavir improved osteoclast activity through the abrogation of a physiological block to RANKL signalling represented by interferon gamma (IFN-γ)-mediated proteosomal degradation of TNF receptor associated factor 6 (TRAF-6) . RANKL recruits signal adapter TRAF-6 to the cytoplasmic tail of RANK resulting in the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPKinase) pathways  involved in the survival and differentiation of osteoclasts. A subsequent paper  showed that ritonavir had opposite effects blocking osteoclastogenesis by impairing RANKL-induced signals. This finding suggested a complex scenario in the interactions between protease inhibitors and the osteoclast lineage. Protease inhibitors were also assayed on the human mesenchymal stem cells differentiating to osteoblast lineage. These experiments indicated that nelfinavir and lopinavir inhibited bone formation and calcium deposition thereby decreasing osteoblast activity . A recent paper by Malizia et al.  assayed four protease inhibitors (nelfinavir, saquinavir, indinavir, ritonavir) on primary osteoblasts and found a significantly decreased osteoblast activity (decrease of alkaline phosphatase, calcium deposition and RUNX-2 mRNA expression) when indinavir and ritonavir were used. Altogether these in-vitro studies suggest that some protease inhibitors may determine bone loss by increasing osteoclast resorption and inhibiting the osteoblast rebuilding function.
It is noteworthy that some in-vitro studies investigated the possible association between use of protease inhibitors and decreased vitamin D serum levels. Vitamin D is essential for the maintenance of a normal bone structure increasing the phosphate bone bioavailability . The biological effects on bone remodelling are exerted by 1,25-dihydroxyvitamin D3 (calcitriol), a potent calcitropic hormone . Vitamin D activation to calcitriol involves 25-hydroxylation in the liver followed by 1α -hydroxylation of 25-hydroxyvitamin D3 in the renal proximal tubular cells, whereas vitamin D catabolism is mainly determined by 24-hydroxylase. The vitamin D deficit can progressively determine osteomalacia through the reduction of phosphates available to bone .
The 1α-hydroxylase and 25-hydroxylase enzymes, involved in vitamin D activation, are cytochrome P450 monoxygenases  and protease inhibitors are potent in-vivo inhibitors of human hepatic cytochrome P450s namely CYP3A4 [113–115]. Ritonavir, but also indinavir and nelfinavir, negatively affect the 1α-hydroxylase enzyme and, to a lesser extent, 25-hydroxylase enzyme activity reducing 1,25-dihydroxyvitamin D3 production (Table 3) whereas no inhibition of 24-hydroxylase is observed [115,116].
Some clinical studies investigated vitamin D deficiency in HIV-infected individuals. Hypovitaminosis D was observed before the advent of HAART and a severe depletion of vitamin D was associated with advanced infection and immune system hyperactivation . Recent studies performed on a cohort of naive and HAART-treated patients demonstrated a high prevalence of hypovitaminosis D suggesting a risk of osteomalacia [118,119]. These results, coupled with the TDF effects on vitamin D regulation indicate that HAART-mediated osteomalacia may be an additional mechanism of bone depletion. Osteomalacia could be underestimated as a pathogenetic event of bone impairment in HIV infection. This problem is not fully appreciated in the literature, because the few studies that have tried to explore bone impairment have usually used the DXA scan. This procedure is a good approach to determine bone mineral content but cannot discriminate between osteoporosis (low BMD and bone architecture deterioration) and osteomalacia (low BMD with normal bone architecture).
In daily clinical practice, doctors must address many problems affecting their HIV-infected patients, such as the prevention of cardiovascular disease, diabetes, dyslipidaemia and lipodystrophy. Although specific complete guidelines are not yet available, several diagnostic approaches have been recommended to monitor bone conditions during HIV disease and the HAART regimen (Table 4).
Hip and lumbar-spine DXA analysis is a valuable approach to determine BMD variations and will discriminate between cortical and trabecular bone, two different compartments that may respond differently to antiretroviral drugs. Hence, DXA should be performed in all patients, at least at baseline visit, as many prospective studies have demonstrated that it will predict fracture risk . A widely cited meta-analysis indicated that the risk of hip fracture increased 2.6-fold for each standard deviation decrease in BMD at the femoral neck . Unfortunately, DXA machines are not widely available and many doctors cannot prescribe the analysis easily, but a major effort should be made to obtain at least one BMD measurement in all HIV-infected patients.
The recent guidelines devised by the International Society for Clinical Densitometry (ISCD) recommend using the T-score with the diagnostic cut-off value specified by WHO only for women in menopause. Although definitive data are lacking, it is generally accepted that the same method can be applied to men over the age of fifty if they have at least one major risk factor for osteoporosis. For individuals aged less than 50 years, diagnosis is recommended using the Z-score that compares the patient's BMD with that of a healthy age-matched and sex-matched population. However, the Z-score has no clear cut-off value for osteopenia and osteoporosis and the following scores are recommended: patients with values lower than −1 are classified as having low bone mass, whereas a severe bone mass reduction is identified by Z-score values lower than −2 . The Z-score may allow further in-depth analysis of BMD and a useful application of this parameter in the evaluation of bone loss in HIV-infected individuals. Osteomalacia can only be diagnosed by bone histomorphometry that will disclose large amounts of unmineralized bone matrix. As bone biopsies are invasive, the diagnosis of osteomalacia is indirect and is generally established by coupling DXA analysis with some blood analytes (i.e. vitamin D, calcium, phosphate, PTH). Hence, DXA alone may underestimate the osteomalacia rate in this population as an unknown number of patients may have been misdiagnosed with osteoporosis. Nonetheless, a correct diagnosis must be established in order to institute appropriate therapy. Osteoporosis is commonly treated with antiresorptive or anabolic agents  whereas osteomalacia requires high doses of vitamin D .
Dorsal and lumbar spine radiograph are useful to assess vertebral fractures. Radiological investigation must be entertained in patients with back pain (particularly occurring in the upright position), patients with marked height reduction and in patients with severe kyphosis; at follow-up spine radiograph must rely upon the physician's decision particularly in patients with a diagnosis of vertebral fractures. Bone biology parameters can be monitored by laboratory tests: calcaemia, phosphataemia and albuminaemia (for ionized calcium calculation), urinary calcium and phosphate excretion. In addition, bone formation markers (i.e. osteocalcin or bone-associated alkaline phosphatase) and bone resorption markers (pyridinoline cross links or CTx, or NTx) can yield useful information together with vitamin D and PTH assays. Important information may be obtained by GFR determination (age and TDF, together with a low GFR, inducing a decrease in the function of alpha-1-hydroxylase in the kidney and in vitamin D availability) and the evaluation of kidney tubular function (some proximal tubular alterations may occur with normal GFR, and may signify an initial impairment of tubular function potentially unsafe for bone). Plasma lactic acidaemia monitoring at baseline and periodically during follow-up may provide valuable information on the side effects of NRTIs on bone. Mora et al.  suggested that plasma RANKL and OPG determination may be employed in some cases of therapy evaluation, as impairment of the RANKL/OPG system is well described in patients receiving protease inhibitor-based HAART. Even though short-term data indicate that replacing stavudine and protease inhibitor with tenofovir and efavirenz restores the RANKL/OPG equilibrium and may thus lead to a reduction in the bone resorption rate. The determination of these cytokines is not yet part of the routine evaluation of HIV-infected patients.
The management of osteopenia/osteoporosis in the course of HIV infection may be based on a change in risk factors, calcium and vitamin D diet supplementation and biphosphonate drugs (Table 5).
Patients can be advised to stop smoking and take physical exercise to control body weight. In addition, patients should have correct diet indications to ensure an adequate uptake of calcium and vitamin D. However, cholecalciferol should be administered to all HIV-infected patients presenting vitamin D deficiency. The daily upper tolerable limit for vitamin D is fixed at 2000 international unit (IU), but this is far below the toxic threshold, that has never been reached even with the administration of 10000 IU per day . A supplementation of 800 IU cholecalciferol daily can be suggested to all outpatients. The only contraindication to cholecalciferol administration is hypercalcaemia and therefore when serum ionized calcium is normal, vitamin D can be used.
A low calcium diet has been demonstrated to reduce BMD and maybe to increase the hip fracture risk [123,124]. The daily calcium recommended allowance for adults is between 800 and 1000 mg per day , but it is very difficult to reach this threshold in cholesterol lowering diets. Therefore, it is very important for correct diet counselling to be given to all HIV-infected patients.
When vitamin D deficiency and low calcium intake have been corrected, drugs can be used. Many treatments are available to combat osteoporosis. The antifracture efficacy, at least for vertebral fractures, is well demonstrated for bisphosphonates (alendronate, risedronate, ibandronate, and zoledronate), hormone replacement therapy, raloxifene, strontium ranelate, teriparatide and recombinant human parathyroid hormone (PTH) .
Four studies have been published on the pharmacological treatment of HIV-induced osteoporosis with alendronate. Due to the low number of individuals analysed, the results obtained have a limited statistical significance, even though they showed that alendronate increased BMD with respect to placebo [126–129]. More recently, similar results were obtained in two trials comparing once yearly zoledronate therapy with placebo [130,131]. No data are available on fracture incidence reduction yet. Further studies are necessary to investigate the effects of bisphosphonates both on BMD and fracture risk reduction in HIV-related bone disease. Nevertheless, especially in fractured patients, the risk of new fractures is very high and therefore this therapeutic approach is mandatory.
The advantages and disadvantages of bisphosphonate treatment must be clearly evaluated for each patient. Notwithstanding the scant data available, alendronate or zoledronate must be considered to enhance bone mineral density and possibly decrease fracture incidence. The adverse effects associated with bisphosphonates include gastrointestinal intolerance (with oral administration), short-lived acute phase reaction (with intravenous administration) and jaw osteonecrosis , but the relatively low risk of this last adverse effect does not contraindicate the use of bisphosphonates at present.
A possible future direction in the treatment of osteoporosis may be the use of the anti-RANKL monoclonal antibody (denosumab). As described earlier, RANKL induces osteoclast activation and its upregulation was noted both in HIV-positive [60–62] and HIV-negative patients with osteopenia/osteoporosis . A clinical study on 412 HIV-negative postmenopausal women given denosumab for 1 year demonstrated an increase in BMD and a decrease of bone turnover . This finding suggested its possible use in osteoporosis treatment even in HIV-positive individuals. Other compounds such as OPG (RANK/RANKL interaction inhibitor), raloxifene (selective estrogen receptor modulator), teriparatide (PTH analogue), PTH and strontium ranelate (dual action bone agent) are under study but, like denosumab (RANK/RANKL interaction inhibitor), further evaluation is needed before these drugs can be administered in HIV-infected patients (Table 6).
Bone derangement is a major clinical complication in the course of HIV infection . The advent of HAART has led to a longer life expectancy and therefore HIV/HAART-related bone disease is destined to increase, enhancing the physiological age-related bone loss. Some epidemiological surveys in HIV-uninfected individuals indicate that the percentage of osteoporosis after 45 years of age ranges between 10 and 20%, and the lifetime fracture risk for a 50-year-old White woman is greater than 50% [136,137]. These data suggest that the number of HIV patients with bone disease and silent fractures can be expected to increase dramatically in the next few years because these patients also have two other potentially worsening factors: HIV itself and antiretroviral therapy. Hence, antiretroviral therapy must be accompanied by the clinical management of HIV/HAART-related bone disease to reduce the risk of osteoporosis and fractures in these patients.
This work was funded by Fondazione Cassa di Risparmio Bologna, Italy (n° 2006.0035, June 2006), ‘AIDS projects’ (30G.27) of the Italian Ministry of Health, the SIVIM study group for test standardization, Funds for selected research topics of the University of Bologna and MURST 60%.
Transparency Declaration: all the authors declare that they have no relationship (commercial or otherwise) that may constitute a dual or conflicting interest.
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