Autologous bone marrow-derived mononuclear cells for therapeutic arteriogenesis in patients with limb ischemia
A double blind placebo controlled study in diabetic and non-diabetic patients

Peripheral arterial occlusive disease (PAD) is a common condition with an
age-adjusted prevalence of approximately 12% in the older population. The
prevalence of PAD increases with age and an estimated 20% of the population
over 70 years is affected. The by far most common symptomatic manifestation of
PAD is mild to moderate claudication which has an age adjusted prevalence of
4.5% in the adult population, but prevalence rapidly increases with age,
resulting in a prevalence rate of over 20% in the population aged 75 years and
older. Owing to the formation of an adequate collateral network, the general
course of claudication is mild, and in the majority of patients symptoms
resolve spontaneously or stabilize with modest impairments in ADL activities.
However, in one quarter of the patients the disease has a less favorable course
and progression of PAD ultimately leads to disabling claudication and critical
leg ischemia, characterized by severe rest-pain and ischemic gangrene. The
greater part of these patients may benefit from intervention procedure(s) (such
as angioplasty and bypass operations) restoring blood supply to the ischemic
area. Unfortunately, in a substantial proportion of patients (i.e. patients
with diabetes mellitus(1) or patients requiring distal repair in the absence of
an adequate vein graft) the prospects of intervention are notably poor, and in
patients with small vessel disease and occlusion of the tibial arteries
intervention may even be impossible. Prognosis for these patients in terms of
quality of life, life expectancy and costs is poor and these patients can only
be treated by a below- or above knee amputation (currently: ±1000/year in The
Netherlands).
Advances in the field of vascular biology have led to concept of stimulation of
collateral formation (or arteriogenesis) in situations in which
revascularization procedures have failed or were not possible. The process of
arteriogenesis is not fully understood but it is now conceived that increased
sheer stress and overstretching of pre-existing interconnecting arterioles lead
to endothelial cell activation and up-regulation of adhesion molecules.
Mononuclear cells adhere to the activated vascular wall, transmigrate into
peri-vascular space and become activated. This process is further amplified by
a perpetual cycle through release of proinflammatory cytokines from activated
monocytes and macrophages. Under the influence of a myriad of released growth
factors, cytokines and possibly bone marrow-derived stem cells the arteriole
undergoes extensive remodeling, which ultimately results in formation of an
artery-like musclized conduit which,
compared to its original size, has increased approximate 40-fold in diameter.(2)
The putative pivotal role of monocytes and macrophages in the process of
arteriogenesis led to the hypothesis that transplantation of BM-MNC may
argument collateral formation in cases where collateral formation is absent or
has failed. This concept was confirmed in several animal models of limb
ischemia,(3,4) which ultimately compiled into a clinical phase I-II study by
the TACT group, published in the Lancet.(5) In this study, Tateishi and
colleagues showed that local injection of BM-MNC in the gastrocnemius muscle of
the ischemic leg was feasible and safe, significantly improved pain free
walking distance, Brachial-Ankle Index, transcutaneous oxygen tension and
reduced pain scores during a 24 week follow up.
Although the beneficial effects

To validate and extend findings from the TACT sytudy group as well as our own
preliminary data in a randomized, placebo controlled study in patients with and
without diabetes.

A single center, randomized placebo controlled study, in patients with
disabling claudication who are not amendable for conventional treatment

Bone marrow will be harvested from the iliac crest (700-800 ml) and the BM-MNC
concentrated to a final volume of 40 mL (average total cell count 1.5-6.0 109
cells). Patients in the placebo group will receive diluted erythrocytes
(hematocrit 5%). This preparation is visually indistinguishable from BM-MNC
concentrate. BM-MNC from the placebo-treated patients will be frozen for later
use (see further). Remaining erythrocytes will be returned to the patients.
As results of our preliminary study indicate that exclusive intra muscular
delivery is equally effective as combined intra arterial/intra muscular
delivery, intra muscular delivery (40 injections in a 3X3 cm grid,
gastrocnemius muscle) will be used as mode of delivery in this study for
practical reasons.
Bone marrow derived mononuclear cells from placebo treated patients will be
frozen and available to the patients upon reaching the primary end-point (t=6
months or eminent amputation).

Harvesting of bone marrow and the intramuscular injections are associated with
significant discomfort, yet application of local-regional anesthesia during the
whole procedere has significantly reduce the burden to the patient. Volume
shiftst during bone marrow may result in cardiac problems, for this reason
patients at risk will remain under close cardiac supervision .