Ultrasonography in Hemophilic Joint Disease and Serum Markers

Background: Hemophilic joint disease secondary to recurrent hemarthroses is one of the most
disabling and costly complications of hemophilia. Prior to widespread use of prophylactic
factor concentrates, children in the United States with severe hemophilia A and B (X-linked
recessive disorders with <1% factor VIII/IX (FVIII/FIX) activity) experienced an average of
30-35 hemarthroses per year reported for FVIII deficiency (3,4 ). Clinical and sub clinical
hemarthroses during childhood results in synovitis (hypertrophied synovium characterized by
villous formation, markedly increased vascularity and chronic inflammatory cells (5)
eventually leading to pannus formation and destructive arthritis (6, 7). At this time,
synovial bleeding may be related not only to clotting factor deficiency but also to
pre-existing vascular damage and inflammation, which is difficult to control clinically. Use
of factor concentrate prophylaxis results in hemophilia patients experiencing fewer joint
bleeds, less rapid deterioration of joint function and fewer days lost from school or work.
However, it may be complicated by the unpredictable development of an inhibitor (a
high-affinity, polyclonal, function-neutralizing antibody directed against FVIII/FIX), with
an incidence of about 25% and 6% respectively (8) . These individuals have limited treatment
options or are treated with products that are less efficacious in treating the joint bleed
along with potential deleterious side effects such as thrombogenecity( 9) thus favoring joint
disease development.

Primary prophylaxis (infusion of FVIII concentrates (25 - 40 u/kg thrice weekly or FIX
concentrates - 80-100u/kg twice a week starting at age 1-2 yrs) before the onset of joint
bleeds is used in Sweden since the 1960s to keep the trough level of factor VIII/FIX > 1%,
converting a severe hemophilia patient (FVIII/FIX activity <1%) into a milder form (FVIII/FIX
>5%) (10 ) . This strategy is expensive (~ $77,760/year for a 20 kg child based on the use of
3000 to 6,000 u/kg/yr with recombinant factor VIII), and may require the use of venous access
devices in young children, which is complicated by severe infections, bleeding and thrombosis
(11) . Secondary prophylaxis on the other hand, which involves the use of FVIII/FIX
concentrates after "target joints" (at least four bleeds occurring into a single joint in the
previous six months) have been identified may limit bleeding and subsequent joint damage.
However, progression of existing joint disease continues and it is unclear whether secondary
prophylaxis can actually prevent joint deterioration (12) . Furthermore, studies comparing
primary and secondary prophylaxis in relation to cost-effectiveness and long-term joint
morbidity suggest that primary prophylaxis improved long-term joint outcome but was twice as
expensive (13 ) . For these reasons, the optimum age, subject population, and timing of
prophylaxis is highly debated. Finally, therapeutic options for individuals who fail or
cannot use prophylaxis (inhibitor patients) or refuse prophylaxis include isotopic (IS) and
surgical synovectomy. Isotopic synovectomy involves intraarticular injection of 32P- colloid
with the intent of scarring off the synovium leading to a subsequent reduction in
hemarthroses (14) , both procedures being recommended for patients with chronic synovitis and
ongoing hemarthroses. Again, the timing of these strategies in relation to the onset of
synovitis remain unclear. Hence, if prophylaxis is not started early (before the occurrence
of joint bleeds) and subject population is not optimized, a strategy to detect and monitor
synovitis and joint arthropathy is urgently needed so that prophylaxis and synovectomy can be
timed based on evidence to reap optimum benefits. Furthermore, in hemophilic children who
complain of joint pains, clinical examination sometimes, may not clearly define whether the
symptoms are related to a joint bleed, synovitis or surrounding soft tissue bleeds. Studies
in animals suggest that cartilage damage can occur concurrently with synovial damage (15)
contributing to joint arthropathy. Therefore, it seems that determination of both synovial
and cartilage changes would be imperative and may help to guide prophylaxis.

Traditionally, hemophilic arthropathy has been diagnosed by clinical examination and plain
radiographs of joints, which together tend to underestimate the extent of joint destruction
(16) . Magnetic Resonance Imaging (MRI) can estimate the degree of bony damage associated
with hemophilic joint disease (14 , 17 -19). The investigators have previously shown the
utility of ultrasound-power Doppler sonography( USG-PDS) in detecting synovitis associated
with hemophilic joint disease when compared to MRI ( 1) . The need for sedation in children
and high costs ($ 2500 for MRI with sedation versus $ 600 for USG- PDS - no sedation at this
institution) override the utility of this tool when repeated studies may be required for
closer surveillance of joint disease progression. Visualization of cartilage is clinically
relevant because benefits of both prophylaxis and synovectomy are realized only if there is
minimal damage to cartilage. Furthermore, there is scattered evidence to suggest that
isotopic synovectomy in a joint affected by bony arthropathy can lead to progression of the
arthropathy leading to crippling arthritis.

The pathogenesis of HJD is not well defined. Neoangiogenesis is a critical factor in
processes, such as tumor growth and inflammatory arthritis (20). Increased vascularity and
neoangiogenesis have been implicated in the progression of musculoskeletal disorders and
tumor growth. Vascular endothelial growth factor (VEGF), the principal signaling molecule in
angiogenesis, can be induced by hypoxia and certain cytokines through interaction with its
receptors, VEGFR1 and VEGFR2 (21 -23). The synovitic pannus in other joint diseases that
share histologic similarities with hemophilic joint disease (HJD) have enhanced oxygen demand
and show evidence of de novo blood vessel formation, including endothelialization of the
synovium( 24) . Further, VEGF expression in the serum has been correlated with disease
activity in rheumatoid arthritis ( 25) . Endothelialization may occur as a result of mature
endothelial cell migration or through the recruitment of bone marrow (BM)-derived endothelial
progenitor cells (EPCs) and hematopoietic progenitor cells (HPCs) from the peripheral
circulation (26) . Importantly, proliferating synovium can secrete chemocytokines, such as
VEGF, that might promote recruitment of endothelial cells (ECs) to sites of active
angiogenesis ( 25) . Co-localization of hypoxia-inducible factor- 1 (HIF-1 α) which is a
transcription factor involved in the induction of VEGF and produced in response to hypoxia
within the joint and VEGF emphasizes the role of hypoxia in the up-regulation of angiogenesis
in rheumatoid joint diseases ( 27).

The investigators have previously observed a 4-fold elevation in proangiogenic factors
(vascular endothelial growth factor-A [VEGF-A], stromal cell-derived factor-1, and matrix
metalloprotease-9) and proangiogenic macrophage/monocyte cells (VEGF+/CD68+ and VEGFR1+/CD11b
+) in the synovium and peripheral blood of hemophilic joint disease (HJD) subjects along with
significantly increased numbers of VEGFR2+/AC133 + endothelial progenitor cells and CD34+/
VEGFR1+ hematopoietic progenitor cells. Sera from HJD subjects induced an angiogenic response
in endothelial cells that was abrogated by blocking VEGF, whereas peripheral blood
mononuclear cells from HJD subjects stimulated synovial cell proliferation, which was blocked
by a humanized anti-VEGF antibody (bevacizumab). Human synovial cells, when incubated with
HJD sera, could elicit up-regulation of HIF-1α mRNA with HIF-1α expression in the synovium of
HJD subjects, implicating hypoxia in the neoangiogenesis process. The investigators results
provided evidence of local and systemic angiogenic response in hemophilic subjects with
recurrent hemarthroses suggesting a potential to develop surrogate biologic markers to
identify the onset and progression of hemophilic synovitis( 2). Therefore, evidence of
increased synovial vascularity on USG-PDS and elevated angiogenic markers suggestive of
increased vascularity in hemophilic joint disease subjects provides a compelling opportunity
to develop surrogate biological markers for hemophilic joint disease. This would also further
aid in tailoring strategies such as prophylaxis and synovectomy in an individual patient.

Inclusion Criteria:

1. All children ages 6 months - 18 years with hemophilia A or B

2. Hemophilia subjects with and without a history of hemarthroses including target joints
( joint of interest) and joints without documented bleeds( control joints)

3. Hemophilia subjects with a history of inhibitor to FVIII or FIX and documented
hemarthroses

4. History of hemarthroses more than 4 weeks prior to study enrolment to allow for
resolution of hemarthroses which could affect detection of synovial and cartilage
changes

Exclusion Criteria:

1. Bleeding disorder subjects without a diagnosis of hemophilia

2. Hemophilia subjects with any underlying illness such as liver or renal disease or any
systemic illness such as diabetes or any other chronic illness apart from the
hemophilia

3. Hemophilia subjects on medications which could increase bleeding risk such as non
steroidal anti inflammatory agents, anti seizure medications apart from factor
concentrates

4. History of hemarthroses within the 4 weeks prior to study enrolment