Highly Active Anti-Retroviral Therapy for HIV-infected individuals impedes bone mineral

Introduction: metabolism and increases prevalence of HAART-related bone disease

Infection with HIV-1, which develops into AIDs
in almost all hosts, has been classified as a human
pandemic for decades, eliciting life-threatening
opportunistic infections to claim the lives of
millions. Advances in anti-retroviral therapies
(ART) have offered HIV-infected persons an
opportunity for prolonged survival. However,
research has shown that extended exposure to the
protease inhibitors (PIs) and nucleoside reverse
transcriptase inhibitors (NRTIs) that compose
ARTs often generate dangerous mitochondrial
toxicities and the alteration of important
transcriptional processes. One prevalent side
effect, among many, from highly active anti-
retroviral therapies (HAARTs) is the pathogenesis
of both osteopenia and osteoporosis.
Figure 2: Osteoclastogenesis
increased in PBMCs in presence of
AZT.
• The effectiveness of AZT using human
peripheral blood mononuclear cells was
observed using HIV+ and HIV- patients.
• PBMCs from 2 HIV-positive, HAART-naïve
patients and HIV-negative individuals were
cultured with RANKL alone (R), or RANKL
plus AZT (R+A) in the presence of M-CSF for
2 weeks. M-CSF alone treated PBMCs served
as a control (C). The number of multinucleated
TRAP positive osteoclasts were counted.
Figure 4: Presence of AZT
results in mitochondrial toxicity
but is reversed by application of
RANKL.
(A) RAW264.7 cells were plated and
treated with AZT and AZT plus
RANKL for 1–3 days.
• Mitochondrial DNA damage
determined by qPCR. Frequency of
mtDNA damage induced by AZT and
AZT+RANKL expressed by
comparison to mtDNA damage in
RANKL-treated cells.
By Tara Manny
Results:
• Osteoclastogenesis is associated with increased activities of
tartrate-resistant acid phosphatase (TRAP) promoter and NF-kB
transcription factor.
• Graph in Figure 1A displays how the osteoclastogenic effect of
AZT is concentration dependent, with less effect above and
below 5.0uL AZT +RANKL.
• Presence of AZT increases expression of TRAP and calcitonin
receptor (CTR), but RANK and GAPDH2 are unaffected
(FIG1B).
• Osteoclastogenesis increases most in the RANKL+AZT sample
in HIV-positive HAART-naïve PBMCs (Fig2).
• Mice treated with AZT showed a reduction in BMD by 6.15 ±
2.02% (Fig3A).
• Histomorphometric analysis shows that mice treated with AZT
had a significant increase in osteoclast surface and number.
• Figure 4A illustrates that RANKL treatment almost completely
reverses mtDNA damages incurred by the presence of AZT.
• Mitochondrial aconitase activity showed a 41% decrease in
RAW264.7 cells treated with AZT from the control. Activity
was reversed or recovered by addition of RANKL, to
approximately 78% of the control (Fig4B).
 Conclusion:
• As levels of HAART increase and are made more readily available to
the HIV positive community, coincidences of osteopenia and
osteoporosis, among other complications, are likely to increase as
well.

Figure 1: Osteoclastogenic effects by AZT
(Zidovudine) in RAW264.7 cells are
apparent.
(A) RAW264.7 cells were treated with AZT at
various concentrations in the presence of 50 ng/mL
RANKL for 3 days. TRAP staining assay gave an
osteoclast measurement, as shown in the graph.
(B) For 1 to 3 days, RAW264.7 cells were treated
with RANKL (50 ng/mL, R) or RANKL plus AZT
(25 μM, R + A) and total mRNAs were isolated.
• (B) Aconitase activity of cell lysates • Over-production of TRAP-positive osteoclasts leads to bone disease.
from RAW264.7 cells treated as
indicated for 3 days • PBMC test results support the conclusion that HIV infection does not
itself accelerate osteoclastogenesis or the effect of AZT in vitro.
• (C) Western blot for mitochondrial
SOD in RAW264.7 cells treated as • AZT induces mtDNA damage while RANKL treatment completely
indicated [for 1–3 days].
reverses mtDNA damage associated with AZT treatment.
• Since increased RANKL levels minimize the deleterious effects of
NRTIs on osteoclast mitochondria, it is possible that they promote
Figure 3: Presence of AZT decreases bone marrow density osteopenia/osteoporosis.
(BMD) and increases number and surface of osteoblasts.
Mice, 5 months old, were orally fed PBS (n = 6, control) or AZT (n 5, AZT 100 • The osteoclastogenic effect of AZT requires the presence of RANKL.
mg/kg/day) for 30 days. Thus, interrupting RANKL by OPG, RANK-Fc, or other compounds
• BMD measured by DEXA. may be potent therapeutic options.
• Histomorphometry was analyzed in three vertebrae of each animal. Osteoclast References:
  Pan, George. "Pathogenesis of Osteopenia/Osteoporosis Induced by Highly Active Anti-Retroviral Therapy for
parameters included number (OcN) and surface (OcS) of osteoclasts. AIDS." Ann N Y Acad Sci. Author manuscript. 1068. (2006): 297-308. Print.
Reported as percentage change using AZT from control.
Pisani, Elizabeth. The Wisdom of Whores. New York: W. W. Norton & Co. Inc., 2008. Print.