U.S. patent application number 11/304865 was filed with the patent office on 2006-11-02 for methods for improving cell therapy and tissue regeneration in patients with cardiovascular and neurological diseases by means of shockwaves.
This patent application is currently assigned to Dornier MedTech System GmbH. Invention is credited to Alexandra Aicher, Stefanie Dimmeler, Harald Eizenhofer, Christopher Heeschen, Andreas Lutz, Andreas Michael Zeiher.
Application Number | 20060246044 11/304865 |
Document ID | / |
Family ID | 35929899 |
Filed Date | 2006-11-02 |
United States Patent
Application |
20060246044 |
Kind Code |
A1 |
Lutz; Andreas ; et
al. |
November 2, 2006 |
Methods for improving cell therapy and tissue regeneration in
patients with cardiovascular and neurological diseases by means of
shockwaves
Abstract
Improving cell therapy and tissue regeneration in a patient
suffering from a cardiovascular or a neurological disease by
treating a tissue of the patient with shock waves and/or applying
to the patient a therapeutically effective amount of stem cells
and/or progenitor cells. Such treatment increases expression of
chemoattractants, pro-angiogenic factors, and pro-survival factors.
The chemoattractants can be, for example, vascular endothelial
growth factor (VEGF) or stromal cell derived factor 1 (SDF-1). For
example, the treated tissue can be located in the patient's heart
or in a skeletal muscle of the patient, and the shock waves can be
extracorporeal shock waves (ESW) or intracorporeal shock waves. The
cardiovascular disease can have an ischemic or non-ischemic
etiology. For example, the cardiovascular disease can be a
myocardial infarction, ischemic cardiomyopathy, or a dilatative
cardiomyopathy. For example, the neurological disease can be a
peripheral neuropathy or neuropathic pain.
Inventors: |
Lutz; Andreas; (Seefeld,
DE) ; Eizenhofer; Harald; (Seefeld, DE) ;
Zeiher; Andreas Michael; (Frankfurt, DE) ; Dimmeler;
Stefanie; (Frankfurt, DE) ; Heeschen;
Christopher; (US) ; Aicher; Alexandra;
(Frankfurt, DE) |
Correspondence
Address: |
KING & SPALDING LLP
1180 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
Dornier MedTech System GmbH
Wessling
DE
|
Family ID: |
35929899 |
Appl. No.: |
11/304865 |
Filed: |
December 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60636204 |
Dec 15, 2004 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
435/440; 435/455; 601/1 |
Current CPC
Class: |
A61K 38/1866 20130101;
A61K 35/44 20130101; A61K 38/195 20130101; A61P 25/04 20180101;
A61K 38/1866 20130101; A61B 17/22004 20130101; A61H 23/008
20130101; A61P 25/00 20180101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61P 21/00 20180101; A61K 35/44
20130101; A61K 38/195 20130101; A61P 9/10 20180101; A61P 43/00
20180101; A61B 2017/00247 20130101; A61P 9/00 20180101; A61B
2018/00392 20130101 |
Class at
Publication: |
424/093.21 ;
435/440; 435/455; 601/001 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61H 1/00 20060101 A61H001/00; C12N 15/09 20060101
C12N015/09 |
Claims
1. A method for improving cell therapy in a patient suffering from
at least one of a cardiovascular disease and a neurological
disease, comprising the step of treating a tissue of the patient
affected by the at least one of the cardiovascular disease and the
neurological disease with shock waves, wherein the tissue is
targeted for cell therapy.
2. The method of claim 1, wherein the patient is affected by the
cardiovascular disease.
3. The method of claim 2, wherein the cardiovascular disease has
one of a non-ischemic etiology and an ischemic etiology.
4. The method of claim 3, wherein the cardiovascular disease is one
of a myocardial infarction, an ischemic cardiomyopathy, and a
dilatative cardiomyopathy.
5. The method of claim 1, wherein the patient is affected by the
neurological disease.
6. The method of claim 5, wherein the neurological disease is one
of a peripheral neuropathy and a neuropathic pain.
7. The method of claim 1, wherein the tissue is located in one of
the patient's heart and a skeletal muscle of the patient.
8. The method of claim 1, further comprising the step of inducing
an expression of at least one chemoattractant factor in the
tissue.
9. The method of claim 8, wherein the at least one chemoattractant
factor comprises one of vascular endothelial growth factor (VEGF)
and stromal cell derived factor 1 (SDF-1).
10. The method of claim 1, wherein the shock waves comprise
extracorporeal shock waves.
11. A method for improving the tissue regeneration in a patient
suffering from at least one of a cardiovascular disease and a
neurological disease, comprising the steps of: treating a tissue of
the patient affected by the at least one of the cardiovascular
disease and the neurological disease with shock waves; and applying
to the patient a therapeutically effective amount of at least one
of stem cells and progenitor cells.
12. The method of claim 11, wherein the patient is affected by the
cardiovascular disease.
13. The method of claim 12, wherein the cardiovascular disease has
one of a non-ischemic etiology and an ischemic etiology.
14. The method of claim 13, wherein the cardiovascular disease is
one of a myocardial infarction, an ischemic cardiomyopathy, and a
dilatative cardiomyopathy.
15. The method of claim 11, wherein the patient is affected by the
neurological disease.
16. The method of claim 15, wherein the neurological disease is one
of a peripheral neuropathy and neuropathic pain.
17. The method of claim 11, wherein the tissue is located in one of
the patient's heart and a skeletal muscle of the patient.
18. The method of claim 11, further comprising the step of
affecting an expression of at least one chemoattractant factor in
the tissue.
19. The method of claim 18, wherein the at least one
chemoattractant factor comprises one of vascular endothelial growth
factor (VEGF) and stromal cell derived factor 1 (SDF-1).
20. The method of claim 11, wherein the shock waves comprise
extracorporeal shock waves.
21. The method of claim 11, wherein the at least one of the stem
cells and the progenitor cells comprise at least one of embryonic
and umbrial cord-blood derived cells.
22. The method of claim 11, wherein the at least one of the stem
cells and the progenitor cells comprise adult cells.
23. The method of claim 22, wherein the at least one of the stem
cells and the progenitor cells are derived from a source selected
from at least one of bone marrow, peripheral blood, and organs.
24. The method of claim 11, wherein the step of applying the
therapeutically effective amount of the at least one of the stem
cells and the progenitor cells comprises the step of applying the
at least one of the stem cells and the progenitor cells by one of
sytemic infusion, local arterial infusion, venous infusion, and
direct injection into the tissue.
25. A method for treating at least one of a cardiovascular disease
and a neurological disease in a patient, comprising the steps of:
treating a tissue of the patient affected by the at least one of
the cardiovascular disease and the neurological disease by means of
shock waves; and applying to the patient a therapeutically
effective amount of at least one of stem cells and progenitor
cells.
26. The method of claim 25, wherein the patient is affected by the
cardiovascular disease.
27. The method of claim 26, wherein the cardiovascular disease has
one of a non-ischemic etiology and an ischemic etiology.
28. The method of claim 27, wherein the cardiovascular disease is
one of a myocardial infarction, an ischemic cardiomyopathy, and a
dilatative cardiomyopathy.
29. The method of claim 25, wherein the patient is affected by the
neurological disease.
30. The method of claim 29, wherein the neurological disease is one
of a peripheral neuropathy and neuropathic pain.
31. The method of claim 25, wherein the tissue is located in one of
the patient's heart and a skeletal muscle of the patient.
32. The method of claim 25, further comprising the step of inducing
an expression of at least one chemoattractant factor in the
tissue.
33. The method of claim 32, wherein the at least one
chemoattractant factor comprises at least one of vascular
endothelial growth factor (VEGF) and stromal cell derived factor 1
(SDF-1).
34. The method of claim 25, wherein the shock waves comprise
extracorporeal shock waves.
35. The method of claim 25, wherein the at least one of the stem
cells and the progenitor cells comprise at least one of embryonic
and umbrial cord-blood derived cells.
36. The method of claim 25, wherein the at least one of the stem
cells and the progenitor cells comprise adult cells.
37. The method of claim 36, wherein the at least one of the stem
cells and the progenitor cells are derived from a source selected
from at least one of bone marrow, peripheral blood, and organs.
38. The method of claim 25, wherein the step of applying the
therapeutically effective amount of the at least one of the stem
cells and the progenitor cells comprises the step of applying the
at least one of the stem cells and the progenitor cells by one of
sytemic infusion, local arterial infusion, venous infusion, and
direct injection into the tissue.
39. A method of using at least one of stem cells and progenitor
cells for preparing a pharmaceutical composition for treating a
patient suffering from at least one of a cardiovascular disease and
a neurological disease, comprising the steps of: subjecting a
tissue of the patient suffering from the at least one of the
cardiovascular disease and the neurological disease to a treatment
with shock waves; and applying to the patient a therapeutically
effective amount of at least one of the stem cells and the
progenitor cells.
40. The method of claim 39, wherein the patient is suffering from
the cardiovascular disease.
41. The method of claim 40, wherein the cardiovascular disease has
one of a non-ischemic etiology and an ischemic etiology.
42. The method of claim 41, wherein the cardiovascular disease is
one of a myocardial infarction, an ischemic cardiomyopathy, and a
dilatative cardiomyopathy.
43. The method of claim 39, wherein the patient is suffering from
the neurological disease.
44. The method of claim 43, wherein the neurological disease is one
of a peripheral neuropathy and a neuropathic pain.
45. The method of claim 39, wherein the tissue is located in one of
the patient's heart and a skeletal muscle of the patient.
46. The method of claim 39, further comprising the step of inducing
the expression of at least one chemoattractant factor in the
tissue.
47. The method of claim 46, wherein the at least one
chemoattractant factor is one of vascular endothelial growth factor
(VEGF) and stromal cell derived factor 1 (SDF-1).
48. The method of claim 39, wherein the shock waves comprise
extracorporeal shock waves.
49. The method of claim 39, wherein the at least one of the stem
cells and the progenitor cells comprise at least one of embryonic
and umbrial cord-blood derived cells.
50. The method of claim 39, wherein the at least one of the stem
cells and the progenitor cells comprise adult cells.
51. The method of claim 50, wherein the at least one of the stem
cells and the progenitor cells are derived from a source selected
from at least one of bone marrow, peripheral blood, and organs.
52. The method of claim 39, wherein the step of applying to the
patient a therapeutically effective amount of the at least one of
the stem cells and the progenitor cells comprises the step of
applying the at least one of the stem cells and the progenitor
cells by one of sytemic infusion, local arterial infusion, venous
infusion, and direct injection into the tissue.
53. The method of claim 39, wherein the step of subjecting a tissue
of the patient with shock waves comprises the step of applying the
shock waves to the tissue before applying to the patient the
therapeutically effective amount of the at least one of the stem
cells and the progenitor cells.
54. The method of claim 39, wherein the step of subjecting a tissue
of the patient with shock waves comprises the step of applying the
shock waves to the tissue while applying to the patient the
therapeutically effective amount of the at least one of the stem
cells and the progenitor cells.
55. The method of claim 39, wherein the step of subjecting a tissue
of the patient with shock waves comprises the step of applying the
shock waves to the tissue after applying to the patient the
therapeutically effective amount of the at least one of the stem
cells and the progenitor cells.
Description
RELATED PATENT APPLICATION
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. Provisional Patent Application No. 60/636,204 filed Dec.
15, 2004 and entitled "Methods for Improving Cell Therapy and
Tissue Regeneration in Patients with Cardiovascular and
Neurological Diseases by Means of Shockwaves." The subject matter
of the above-identified priority application is hereby fully
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for improving cell
therapy in a patient. More specifically, the present invention
relates to methods for improving cell therapy in patient who is
suffering from a cardiovascular or a neurological disease by using
shock waves as a therapeutic tool for targeting the recruitment of
stem cells and/or progenitor cells to a tissue of the patient.
BACKGROUND OF THE INVENTION
[0003] Stem and progenitor cells derived from the bone marrow may
play a role in ongoing endothelial repair (Kalka et al., 2000).
Impaired mobilization or depletion of these cells may contribute to
endothelial dysfunction and cardiovascular disease progression.
Indeed, in healthy men, levels of circulating progenitor cells may
be a surrogate biologic marker for vascular function and cumulative
cardiovascular risk. Recent advances in basic science have also
established a fundamental role for endothelial stem and progenitor
cells in postnatal neovascularization and cardiac regeneration.
Improvement of neovascularization after critical ischemia is an
important therapeutic option after myocardial infarction or limb
ischemia. Until recently, neovascularization of ischemic tissue in
the adult was believed to be restricted to migration and
proliferation of mature endothelial cells, a process termed
"angiogenesis". Meanwhile, increasing evidence suggests that
circulating stem and progenitor cells home to sites of ischemia and
contribute to the formation of new blood vessels. In analogy to the
embryonic development of blood vessels from primitive endothelial
progenitors (angioblasts), this process is referred to as
"vasculogenesis". The importance of circulating stem and progenitor
cells is demonstrated by the fact that genetic inhibition of their
recruitment inhibits tumor angiogenesis. Stem and progenitor cells
can be mobilized from the bone marrow into the circulation by
vascular endothelial growth factor ("VEGF") or stromal cell-derived
factor ("SDF-1"). Both VEGF and SDF-1 are profoundly up-regulated
in hypoxic tissue suggesting that VEGF and SDF-1 may constitute
homing signals to recruit circulating stem and progenitor cells to
enhance endogenous repair mechanisms after critical ischemia.
[0004] The present inventors have recently shown that infusion of
bone marrow mononuclear cells derived from patients with ischemic
heart disease is significantly less effective in improving
perfusion of ischemic tissue in a hind limb ischemia animal model.
Moreover, bone marrow cells of patients with ischemic heart disease
reveal a reduced colony forming activity and an impairment of
migratory response towards VEGF and SDF-1, which are potent
chemoattractive and mobilizing agents (Heeschen C et al.,
Circulation 2004; 109(13): 1615-22.) Moreover, the present
inventors were also able to demonstrate that in experimental models
of tissue ischemia, recruitment of systemically infused
stem/progenitor cells is significantly lower as compared to the
recruitment of stem/progenitor cells derived from healthy donors.
While in patients with acute coronary syndromes, the present
inventors have observed a marked increase in systemic VEGF levels
within 10 hours after onset of symptoms (Heeschen et al.
Circulation 2003; 107(4):524-30), in another set of patients with
acute myocardial infarctions, systemic VEGF levels three days after
the acute event had already decreased and did not significantly
differ from levels measured in patients without coronary heart
disease (Lee et al. NEJM 2000; 342:626-33). Taken together, these
data suggest that, in patients with chronic tissue damage such as
old myocardial infarction, the recruitment of stem/progenitor cells
will be markedly reduced due to the low expression of
chemoattractant factors in the target tissue as well as due to the
low functional activity of autologous stem/progenitor cells from
patients with cardiovascular risk factors.
[0005] Extracorporeal shock waves ("ESW") are generated by high
voltage spark discharge under water. This causes an explosive
evaporation of water, producing high energy acoustic waves. By
focusing the acoustic waves with a semi-ellipsoid reflector, the
waves can be transmitted to a specific tissue site (Ogden et al.,
2001). ESW have been found beneficial in certain orthopedic
conditions. The interactions of ESW with the targeted tissue are
manifold: mechanical forces at tissue interfaces related to
different acoustic impedances, as well as micro-jets of collapsing
cavitation bubbles are the primary effects. However, the cellular
and biochemical mechanisms, by which these physical effects may
enhance healing of fractures, remain to be determined. It has been
scintigraphically and sonographically implicated that local blood
flow and metabolism of bone and Achilles tendon are positively
affected by ESW treatment (Maier et al., 2002).
[0006] ESW therapy has shown to be effective in the treatment of
orthopedic conditions including non-union of long bone fracture,
calcifying tendonitis of the shoulder, lateral epicondylitis of the
elbow, proximal plantar fasciitis, and Achilles tendonitis (Kruger
et al., 2002). The success of shock wave therapy ranges from 80%
for non-unions of long bone fractures to 15-90% for tendinopathies
of the shoulder, elbow and heel. In addition, the short-term
results of shock wave therapy for avascular necrosis of the femoral
head appear encouraging. Shock wave therapy also showed a positive
effect in promoting bone healing in animal experiments. Despite the
success in clinical application, the exact mechanism of shock wave
therapy remains unknown. Recent experiments in dogs demonstrated,
however, that shock wave therapy enhanced neovascularization at the
tendon-bone junction (Wang et al., 2002). It was hypothesized that
shock wave therapy may have the potential to induce the ingrowth of
new blood vessels and improvement of blood supply that lead to
tissue regeneration. Indeed, a recent study in rabbits showed that
shock wave therapy induces the ingrowth of neovessels and tissue
proliferation associated with the early release of
angiogenesis-related factors including endothelial nitric oxide
synthase (eNOS) and VEGF at the tendon-bone junction in rabbits
(Wang et al., 2003). Therefore, the mechanism of shock wave therapy
may involve the early release of angiogenic growth factors and
subsequent induction of cell proliferation and formation of
neovessels at the tendon-bone junction. The occurrence of
neovascularization may lead to the improvement of blood supply and
play a role in tissue regeneration at the tendon-bone junction.
[0007] It was also reported that the ESW-induced VEGF-A elevation
in human osteoblasts is mediated by Ras-induced superoxide and
ERK-dependent HIF-1 activation.
[0008] Further, it has been demonstrated that ESW enhance
osteogenic differentiation of mesenchymal stem cells in vitro as
well as bone union of segmental defect in vivo through
superoxide-mediated signal transduction (Wang et al., 2002a). These
data indicate that the microenvironment of the defect is indeed
responsive to physical ESW stimulation. Subsequent experimental
studies demonstrated that mesenchymal stem cells adjacent to the
segmental defect were subject to three consecutive events after ESW
treatment: intensive recruitment, proliferation, and chondrogenic
as well as osteogenic differentiation (Chen et al., 2004). The
utilized energy for ESW treatment (0.16 mJ/mm.sup.2 EFD) did not
induce side effects in rats. A major limitation of this in vivo
study is that the morphological techniques utilized for the
identification of mesenchymal stem cells lack specificity. Only few
other studies of bone repair have monitored mesenchymal stem cells
of rats, as specific markers for such cells are scarce.
[0009] Regarding the use of ESW for treating tissues other than
bone, it was shown that ESW therapy ameliorates ischemia-induced
myocardial dysfunction in pigs in vivo (Nishida et al., 2004).
[0010] It is noted that no prior art exists which discloses or
suggests a possible link between ESW therapy and the use of stem
and progenitor cells for cell therapy.
[0011] In summary, post infarction heart failure remains a major
cause of morbidity and mortality in patients with coronary heart
disease. Although prompt reperfusion of the occluded artery has
significantly reduced early mortality rates, ventricular remodeling
processes characterized by progressive expansion of the infarct
area and dilation of the left ventricular cavity result in the
development of heart failure in a sizeable fraction of patients
surviving an acute myocardial infarction. The major goal to reverse
remodeling would be the stimulation of neovascularization as well
as the enhancement of regeneration of cardiac myocytes within the
infarct area.
[0012] Peripheral neuropathy describes damage to the peripheral
nerves. It may be caused by diseases of the nerves or as the result
of systemic illnesses. Many neuropathies have well-defined causes
such as diabetes, uremia, AIDS, or nutritional deficiencies. In
fact, diabetes is one of the most common causes of peripheral
neuropathy. Other causes include mechanical pressure such as
compression or entrapment, direct trauma, fracture or dislocated
bones; pressure involving the superficial nerves (ulna, radial, or
peroneal); and vascular or collagen disorders such as
atherosclerosis, systemic lupus erythematosus, scleroderma, and
rheumatoid arthritis. Although the causes of peripheral neuropathy
are diverse, they produce common symptoms including weakness,
numbness, paresthesia (abnormal sensations such as burning,
tickling, pricking or tingling) and pain in the arms, hands, legs
and/or feet. A large number of cases are of unknown cause.
[0013] Therapy for peripheral neuropathy differs depending on the
cause. For example, therapy for peripheral neuropathy caused by
diabetes involves control of the diabetes. In entrapment or
compression neuropathy, treatment may consist of splinting or
surgical decompression of the ulnar or median nerves. Peroneal and
radial compression neuropathies may require avoidance of pressure.
Physical therapy and/or splints may be useful in preventing
contractures (a condition in which shortened muscles around joints
cause abnormal and sometimes painful positioning of the
joints).
[0014] Ischemic peripheral neuropathy is a frequent, irreversible
complication of lower extremity vascular insufficiency. It has been
shown that ischemic peripheral neuropathy can be prevented and/or
reversed by gene transfer of an endothelial cell mitogen (e.g.
VEGF) designed to promote therapeutic angiogenesis (Schratzberger
P, et al.). The major goal to reverse vascular insufficiency would
thus be the stimulation of angiogenesis and the regeneration of the
vascular tissue within the area affect by peripheral
neuropathy.
[0015] The technical problem underlying the present invention in
thus to enhance the cell therapy and regeneration of tissues
affected by a cardiovascular or a neurological disease.
[0016] According to the invention, this problem is solved by the
provision of a method for improving cell therapy in a patient
suffering from a cardiovascular disease or a neurological disease
comprising a treatment by means of shock waves of an tissue of the
patient affected by the disease, which tissue is targeted for cell
therapy.
SUMMARY OF THE INVENTION
[0017] The present invention provides, in part, a therapeutic tool
improving the targeted recruitment of stem and progenitor cells in
patients undergoing cell therapy.
[0018] The present invention relates to methods for improving cell
therapy in a patient who is suffering from a cardiovascular or a
neurological disease and is undergoing cell therapy by using shock
waves as a therapeutic tool for targeting the recruitment of stem
cells and/or progenitor cells to a tissue of the patient. The
present invention also relates to methods for improving tissue
regeneration in a patient suffering from a cardiovascular or
neurological disease by treating a tissue of the patient affected
by the disease using shock waves. Also provided are methods for
treating a cardiovascular or neurological disease in a patient
comprising the treatment of a tissue of the patient affected by the
disease by means of shock waves, and applying to the patient a
therapeutically effective amount of stem cells and/or progenitor
cells. The present invention further also relates to the use of
stem cells and/or progenitor cells for preparing a pharmaceutical
composition for treating a patient suffering from a cardiovascular
disease or a neurological disease, wherein the patient is subjected
to a treatment with shock waves before, during, or after
administration of the stem cells and/or progenitor cells.
[0019] The present inventors have recently shown that autologous
stem and progenitor cells in patients with cardiovascular risk
factors have a reduced ability to home and migrate to damaged
tissue. Since the expression of chemoattractant factors in
chronically injured tissue is markedly reduced as compared to acute
injury, the overall recruitment of stem/progenitor cells in
patients with cardiovascular risk factors is impaired. The
invention involves the treatment of tissue that is targeted for
therapy with stem and progenitor cells by means of shock waves to
increase the expression of chemoattractants (i.e. factors mediating
the attraction of circulating stem and progenitor cells, e.g.
SDF-1.alpha., VEGF, P1GF) and pro-angiogenic factors (i.e. factors
stimulating pre-existing endothelial cells to form new vessels,
e.g. HIF-1.alpha., VEGF, P1GF) as well as pro-survival factors
(i.e. factors inhibiting apoptosis/programmed cell death, e.g. HGF,
IGF, VEGF). The increased expression of chemoattractant and
pro-angiogenic factors will improve the recruitment of systemically
infused stem and/or progenitor cells, and enhanced expression of
pro-survival factors will improve the microenvironment for cells
directly administered into the target tissue. The homing of stem
and progenitor cells will be enhanced. Thereby, shock wave
treatment of the targeted tissue will enhance the therapeutic
effect of cell therapy.
[0020] By combining the application of extracorporeal shock waves
("ESW") and the application of stem cells and/or progenitor cells,
the regeneration of cardiovascular and neurological diseases may be
improved. The combination of ESW and the application of stem cells
and/or progenitor cells may be used to treat cardiovascular and
neurological diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0022] FIG. 1 is a block diagram depicting shock wave-induced
vascular endothelial growth factor ("VEGF") expression in rat
hindlimb muscles detected by Western blot, according to an
exemplary embodiment of the invention.
[0023] FIG. 2A is a representative image of shock wave-induced VEGF
expression in rat hind limb muscles detected by VEGF staining on
frozen sections, according to an exemplary embodiment of the
invention.
[0024] FIG. 2B is a block diagram depicting shock wave-induced VEGF
expression in rat hind limb muscles detected by VEGF staining on
frozen sections, according to an exemplary embodiment of the
invention.
[0025] FIG. 3A is a representative image of detection of
intravenously injected endothelial progenitor cells ("EPCs") after
shock wave treatment, wherein 10-.mu.m frozen sections were
analyzed for EPCs (red fluorescence) and nuclei were stained with
Tropro-3 (blue fluorescence), according to an exemplary embodiment
of the invention.
[0026] FIG. 3B is a block diagram depicting quantification of
intravenously injected EPCs that were recruited to shock wave
treated muscles, according to an exemplary embodiment of the
invention.
[0027] FIG. 4A is a series of representative images of the ischemic
(left) and non-ischemic (right) limb for animals that received
either no treatment, EPC infusion only, shock wave pretreatment
only, or both, according to an exemplary embodiment of the
invention.
[0028] FIG. 4B is a block diagram depicting quantitative perfusion
data generated by calculating the ratio of the perfusion of the
ischemic to the non-ischemic limb, according to an exemplary
embodiment of the invention.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0029] In one aspect, the invention is related to a method for
improving cell therapy in a patient suffering from a cardiovascular
disease or a neurological disease comprising a treatment by means
of shock waves of a tissue of the patient affected by the disease,
which tissue is targeted for cell therapy.
[0030] In another aspect, the invention relates to a method for
improving the tissue regeneration in a patient suffering from a
cardiovascular disease or a neurological disease comprising the
steps of treating a tissue of the patient affected by the disease
by means of shock waves, and applying to the patient a
therapeutically effective amount of stem cells and/or progenitor
cells.
[0031] In yet another aspect, the invention is related to a method
for treating a cardiovascular disease or a neurological disease in
a patient comprising the steps of treating a tissue of the patient
affected by the disease by means of shock waves, and applying to
the patient a therapeutically effective amount of stem cells and/or
progenitor cells.
[0032] In a preferred embodiment of the methods according to the
invention, the treatment of the patient by shock waves is carried
out prior to the administration of the stem and/or progenitor
cells. However, a simultaneous application of both shock waves and
stem/progenitor cells and a subsequent application of shock waves
(following the administration of the stem/progenitor cells) is also
contemplated.
[0033] In a further aspect, the invention relates to a use of stem
and/or progenitor cells for preparing a pharmaceutical composition
for treating a patient suffering from a cardiovascular disease or
neurological disease, wherein the patient is subjected to a
treatment with shock waves before, during, or after administration
of the stem and/or progenitor cells.
[0034] The term "cell therapy" refers to the transplantation of
cells to replace or repair damaged tissue and/or cells. Cell
therapy involves the use of blood transfusions and bone marrow
transplants, as well as injections of cellular materials.
[0035] Within the meaning of the present invention, the term "shock
waves" is used interchangeably with the term "acoustical pressure
pulse".
[0036] The term "stem cell" refers to an unspecialised cell that is
capable of replicating or self-renewing itself and developing into
specialized cells of a variety of cell types. The product of a stem
cell undergoing division is at least one additional cell that has
the same capabilities as the original cell. The term "stem cell" is
intended to encompass embryonal and adult stem cells, totipotent
and pluripotent cells, and autologous cells, as well as
heterologous cells.
[0037] The definition of "progenitor cell" (also known as a
precursor cell) is intended to encompass cells which are yet
undifferentiated but may already be committed to a specific cell
type (e.g. endothelial progenitor cells are committed to
differentiate into endothelial cells).
[0038] In one embodiment of the invention, the patient's disease is
a cardiovascular disease. In a specific embodiment, the
cardiovascular disease has a non-ischemic etiology. An example of a
cardiovascular disease with a non-ischemic etiology which can be
treated by the methods according to the invention is dilatative
cardiomyopathy. Alternatively, the cardiovascular disease may have
an ischemic etiology. Cardiovascular diseases with an ischemic
etiology which may be improved by cell therapy include myocardial
infarctions and ischemic cardiomyopathies. Chronic ischemic
cardiomyopathy is particularly preferred.
[0039] In another embodiment of the invention, the patient's
disease is a neurological disease. In a preferred embodiment, the
neurological disease is peripheral neuropathy or neuropathic
pain.
[0040] Thus, preferably, the affected tissue is located in the
heart or in a skeletal muscle.
[0041] In a further embodiment of the invention, the expression of
at least one chemoattractant factor is induced in the affected
tissue of the patient. The term "chemoattractant factor" is used
herein to refer to a factor activating the movement of individual
cells, in response to a chemical concentration gradient.
[0042] Preferably, the at least one chemoattractant factor is
vascular endothelial growth factor, VEGF, or stromal cell derived
factor 1, SDF-1.
[0043] The shock waves used in the methods and uses according to
the invention preferably are extracorporeal shock waves which may,
for instance, be applied extra-thoracal. However, also
intracorporeal shock waves (delivered e.g. trans-esophageal) and
endoscopic shock waves (delivered e.g. intraluminal such as in the
artery) are contemplated. Moreover, the shock waves may be applied
during open surgery (intra-operative).
[0044] In a preferred embodiment, 50, 100, 150, or 200 shocks per
area and/or a total number of 100, 250, 500, 1000, or 1500 shocks
per treatment are applied. Preferably, shocks with an energy of
0.05, 0.09, 0.13; 0.22, 0.36, or 0.50 mJ/mm.sup.2 are applied. The
shock waves may be applied once or several times prior to cell
therapy; an application once or twice prior to cell therapy is
preferred. Preferably, the shock waves are applied several hours
before the start of the cell therapy; an application 24 h, 36 h, or
48 h prior to cell injection is particularly preferred.
Alternatively, the shock waves may be exclusively or additionally
be applied during cell therapy and/or after the start of the cell
therapy.
[0045] In one embodiment, the stem and/or progenitor cells which
are used in the methods and uses according to the invention are
embryonic or umbilical cord-blood derived cells.
[0046] Alternatively, the stem and/or progenitor cells are adult
cells. Adult stem and/or progenitor cells can be derived from bone
marrow, peripheral blood, and organs. For example, the cells can be
derived from healthy donors or patients suffering from coronary
heart disease.
[0047] For use in the methods and uses according to the present
invention, the stem and/or progenitor cells are isolated and,
optionally, cultivated ex vivo before being applied.
[0048] In specific embodiments, the following stem and/or
progenitor cells may be used in the methods and uses according to
the invention:
[0049] CD34+CD133+bone marrow-derived stem cells
[0050] CD34+CD38-bone marrow-derived stem cells
[0051] CD34+CD45+bone marrow-derived progenitor cells
[0052] CD34+KDR+bone marrow-derived endothelial progenitor
cells
[0053] CD34-CD45-bone marrow-derived mesenchymal stem cells
(MSC)
[0054] eNOS+KDR+CD105+VE-Cadherin+vWF+CD45+ peripheral
blood-derived endothelial progenitor cells
[0055] stage-specific embryonic antigen, SSEA-4+Oct4+ embryonic
stem cells
[0056] CD34+CD133+ cord blood-derived stem cells
[0057] CD34+CD45+ cord blood-derived stem cells.
[0058] The stem and/or progenitor cells used in the methods and
uses according to the invention may be applied by way of systemic
infusion, local arterial infusion, venous infusion, and/or by
direct injection into the affected tissue. For the purpose of
delivery, the cells may further be encapsulated in microspheres
(targeted drug delivery). Contrast agents used for ultrasound are
examples for useful encapsulation agents. The cells may then be
released from the microspheres at the targeted tissue using
ultrasound (acoustic energy).
[0059] Recent data suggests that, in patients with chronic tissue
damage, such as old myocardial infarction, the recruitment of
stem/progenitor cells is markedly reduced due to the low expression
of chemoattractant factors in the affected tissue. However,
treatment of the targeted tissue by single or repetitive exposure
to shock waves will (re)induce the expression of pro-angiogenic,
chemoattractant, and pro-survival factors such as VEGF and SDF-1
and, thereby, will enhance the recruitment of stem/progenitor
cells. Since the treatment effect of cell therapy is directly
proportional to the number of recruited cells, this enhanced
recruitment and survival of stem/progenitor cells after treatment
with shock waves will increase the therapeutic benefit that the
individual patients will derive from cell therapy for tissue
regeneration and tissue.
[0060] A prerequisite for the success of cell therapy is the homing
and, thus, engraftment of transplanted cells into the target area,
especially if an intravascular route of administration is chosen.
The present inventors have now shown that the migratory capacity of
adult progenitor cells towards their physiological chemo-attractant
reflects their homing capacity into the ischemic/infarcted area.
Indeed, the experimental studies conducted by the present inventors
which are shown in the present invention demonstrate that, in the
hind limb ischemia model of nude mice, homing of transplanted cells
to the ischemic tissue and improvement of neovascularization
induced by intravenous infusion of human progenitor cells closely
correlates with SDF-1-induced migratory capacity for bone
marrow-derived cells, as well as with VEGF-induced migratory
capacity for blood-derived progenitor cells, respectively.
Functional impairment of stem and progenitor cells from aged
individuals and patients with cardiovascular diseases, as well as
the reduced expression of pro-angiogenic, chemoattractant, and
pro-survival factors in the targeted tissue may limit the
beneficial effects of clinical cell therapy. As shown in the
present examples, treatment of the targeted tissue by means of
shock waves will facilitate stem and progenitor recruitment and
survival and, thus, will enhance the therapeutic effect of cell
therapy.
[0061] Specifically, the present inventors identified that:
[0062] a. enhancing the recruitment of stem/progenitor cells is a
novel target for improving the clinical outcome after autologous
cell therapy in aged individuals and patients with cardiovascular
risk factors;
[0063] b. a high level of expression of pro-angiogenic,
chemoattractant and pro-survival factors can be restored by
treatment of the target tissue with shock waves;
[0064] c. treatment of chronically injured tissue by single or
repetitive administration of shock wave prior to autologous cell
therapy improves the clinical outcome after cell therapy.
[0065] The following Figures and Examples are intended for
illustration of the present invention only, and should not be
construed as limiting the scope of the invention.
EXAMPLES
[0066] 1. Materials for Preparing Endothelial Progenitor Cells from
Peripheral Blood
[0067] As the starting material for preparing endothelial
progenitor cells, peripheral blood was freshly drawn and collected
in heparin monovettes (10 ml).
[0068] The following materials were used in the examples described
below.
[0069] Dulbecco's Phosphate Buffered Saline without calcium and
magnesium (Cat. No. H-15-002) was used for suspension of the cells
for injection. PAA was purchased from Laboratories GmbH (Pasching,
Austria). EGM Bullet Kit (EBM medium) (Cat. No. CC-3124) and
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled
acetylated low-density lipoprotein (Dil-Ac-LDL) (Cat. No. #4003)
was obtained from CellSystems (St. Katharinen, Germany). Fetal
Bovine Serum (Cat. No. 10270-106) was obtained form Invitrogen GmbH
(Karlsruhe, Germany). Biocoll Separating Solution, Density: 1.077
(Cat. No. L6115) was purchased from Biochrom AG (Berlin, Germany).
Human fibronectin, 1 mg/ml (Cat. No. F-0895) and lectin from Ulex
europaeus (Cat. No. L-9006) was purchased Sigma (Taufkirchen,
Germany). Human recombinant vascular endothelial growth factor
(VEGF) (Cat. No. 100-20) was acquired from Cell Concepts (Umkirch,
Germany). EDTA disodium salt dihydrate (Cat. No. A1104) was
obtained from AppliChem (Darmstadt, Germany). TURK'S solution (Cat.
No. 1.09277.0100) was purchased from Merck (Darmstadt,
Germany).
[0070] 2. Cell Preparation
[0071] 2.1 Isolation of Endothelial Progenitor Cells from
Peripheral Blood
[0072] Mononuclear cells (MNC) are separated from freshly collected
peripheral blood or buffy coats from the blood donation center
using Ficoll gradient centrifugation. First, 15 ml Biocoll
separation solution are provided per 50 ml tube. The peripheral
blood (PB) is diluted with PBS (PB 1:1 or buffy coats 1:4).
Carefully and slowly, 25 ml of the diluted blood are overlayed on
15 ml of the Biocoll separation solution. The tube is centrifugated
at 800.times.g for 20 min at room temperature without brake. This
is an important step to separate the mononuclear cells (in the
interphase) from erythrocytes and granulocytes (pellet) and
platelets in the upper serum phase. Meanwhile, wells are coated
with 10 .mu.g/ml human fibronectin in PBS, and the wells are
incubated for at least 30 min at room temperature. The mononuclear
cells are pipetted from the interphase carefully in a new 50 ml
tube. PBS is added to 50 ml to wash the cells. The cells were
centrifugated at 800.times.g for 10 min at room temperature (with
brake). The supernatant was removed and the cell pellet was
resuspended in 50 ml PBS. The cells were centrifugated at
800.times.g for 10 min at room temperature (with brake), the
supernatant was removed and the cell pellet was resuspended in 10
ml PBS. An aliquot of the cells (50 .mu.l) was diluted (1:10) with
TURK'S solution and counted. PBS was added to the remaining cells
in the 50 ml tube to wash the cells again, following centrifugation
at 800.times.g, 10 min, room temperature, with brake. The washing
steps should be performed for at least 3 times, but should be
repeated until the supernatant becomes clear (altogether 3-5
times).
[0073] Then, the supernatant is removed and the cell pellet is
resuspended in culture medium (endothelial basal medium
supplemented with 20% FBS, epidermal growth factor (10 .mu.g/mL),
bovine brain extract (3 .mu.g/mL), gentamicin (50 .mu.g/mL),
hydrocortisone (1 .mu.g/mL), VEGF (100 ng/ml) to a cell
concentration of 8.times.10.sup.6 cells/ml medium. Fibronectin is
then removed from the dishes. Next, the cells are added to the
fibronectin-coated wells at a density of approx. 2.1.times.10.sup.6
cells/cm.sup.2 (per 24 well plate: 4.times.10.sup.6 cells in 500
.mu.l medium per well; per 12 well plate: 8.times.10.sup.6 cells in
1 ml medium per well; per 6 well plate: 20.times.10.sup.6 cells in
2.5 ml medium per well). The cells are incubated for 3 days at
37.degree. C. and 5% CO.sub.2. Three days after cultivation, the
non-adherent cells were removed by thoroughly washing the cells
with PBS. Fresh culture medium was added for 24 h before starting
the experiments. Approximately 0.5-1% of the initially applied
mononuclear cells becomes adherent endothelial progenitor cells
(EPCs).
[0074] 2.2. Labeling with Red Fluorescent Cell Tracker CM-Dil
[0075] EPCs were washed with PBS, trypsinized for 2 min, then the
reaction was stopped with serum-containing RPMI medium. Detached
EPCs were washed again with PBS, incubated with CM-Dil (Molecular
Probes) diluted in PBS (1:100) for 5 min at 37.degree. C., followed
by incubation for 15 min on ice. After washing, 1.times.10.sup.6
CM-Dil-labeled EPCs were injected into the jugular vein of nude
rats pre-treated with shock wave therapy.
[0076] 3. Application of Shock Wave Treatment
[0077] Shock waves were applied at graded doses of flux density
(0.13-0.64 mJ/mm.sup.2; 3 Hz; 500 impulses) to the upper right hind
limb of nude rats. The energy was focused on the upper limb, while
moving the focus distally for 2 mm after every 100 impulses.
[0078] 3.1 Shock Wave Treatment to Upregulate Chemoattractant
Factors in the Rat Limb
[0079] To assess whether shock wave treatment up-regulates
pro-angiogeneic growth factors such as VEGF, which is
chemoattractant for VEGF receptor 1 or 2 positive stem and
progenitor cells that are injected after 24 h, shock wave treatment
was performed. The right hind limb of nude rats was treated with a
flux density of 0.13, 0.22, 0.43, and 0.64 mJ/mm.sup.2 (FIG. 1).
The left hind limb was used as a negative control (0 mJ/mm.sup.2).
After 24 h, the shock wave-induced up-regulation of VEGF protein
expression was analyzed in the treated versus the untreated hind
limb by means of Western blotting. It was found that flux densities
up to 0.43 mJ/mm.sup.2 yielded favorable VEGF protein expression
ratios between shock wave-treated versus untreated limbs, resulting
in at least 2-fold induction of VEGF protein expression. The best
ratio (more than 2-fold induction) was obtained by using 0.22
mJ/mm.sup.2. Flux densities higher than 0.64 mJ/mm.sup.2 also
strongly enhanced background levels of VEGF protein expression so
that insufficient ratios were obtained to induce treatment-specific
VEGF protein induction. These data suggest that shock wave
treatment should not be applied over a treshold value to avoid
unspecific VEGF protein induction of the contralateral hind
limb.
[0080] Animal model. Immunodeficient female nude rats (5 to 7-wk
old) underwent shock wave treatment with a flux density of 0.13,
0.22, 0.43, and 0.64 mJ/mm.sup.2, which was delivered to the right
hind limb. The contralateral left hind limb did not receive shock
wave treatment. Twenty-four hours later, rats were sacrificed and
the adductor muscle of the right and left hind limbs were removed,
frozen in liquid nitrogen, and minced in a mortar using 1 ml
protein lysis buffer (20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1
mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium
pyrophosphate, 1 mmol/L .beta.-glycerophosphate, 1 mmol/L Na3VO4, 1
.mu.g/mL leupeptin and 1 mmol/L phenylmethylsulfonyl fluoride) for
15 min on ice.
[0081] Western blot analysis. Proteins (40 .mu.g/lane) were loaded
onto SDS-polyacrylamide gels and blotted onto PVDF membranes. After
blocking with 3% bovine serum albumin (BSA) at room temperature for
2 h, the anti-rat VEGF antibody (R&D, Germany) was incubated in
TBS (50 mM Tris/HCl, pH 8; 150 mM NaCl, 2.5 mM KCl)/0.1%
Tween-20/3% BSA for 2 h. Enhanced chemiluminescence was performed
according to the instructions of the manufacturer (Amersham,
Germany). Then, the blots were reprobed with the ERK antibody
(Biolabs, Schwalbach, Germany) as a loading control. The
autoradiographies were scanned and semiquantitatively analyzed.
[0082] To evaluate VEGF protein expression in tissue sections of
the right hind limb of shock wave-treated nude rats, cryosections
of shock wave-treated versus untreated hind limb muscles were
stained for VEGF expression (FIG. 2a). VEGF expression was detected
as cytoplasmic and secreted VEGF staining (green fluorescence) with
respect to nuclear staining (blue fluorescence). Since flux
densities higher than 0.43 mJ/mm.sup.2 led to high unspecific
background levels of VEGF expression, only flux densities between
0.13 mJ/mm.sup.2 and 0.43 mJ/mm.sup.2 were used (FIG. 2b). Similar
to the VEGF expression ratios obtained by Western blotting, flux
densities of 0.22 mJ/mm.sup.2 induced a more than 2-fold induction
of VEGF protein expression compared with flux densities of 0.13
mJ/mm.sup.2 resulting in lower ratios. In contrast to the results
obtained by Western blotting, the best ratio was obtained by 0.43
mJ/mm.sup.2.
[0083] Histological analysis. Tissue samples of nude rats treated
with or without shock waves as described above and were harvested
after 24 h and frozen in liquid nitrogen pre-chilled with
2-methylbutane in OCT (TissueTec, Sakura, The Netherlands).
10-.mu.m sections were cut and immunostaining was performed.
Anti-rat VEGF (R&D, Wiesbaden, Germany) was directly labeled
with Alexa488 (green fluorescence) using Alexa Fluor R 488 antibody
labeling kit (Molecular Probes, Eugene, Oreg., USA). Nuclear
staining was performed using Topro-3 (Molecular Probes).
[0084] For the quantification of shock wave-induced VEGF expression
in rat hind limb muscles (M. adductor and M. semimembraneous), the
number of VEGF.sup.+ cells per high power (HP) view was determined
for 0.13, 0.22, and 0.43 mJ/mm.sup.2.
[0085] 3.2 Shock Wave-Faciliated Recruitment of Systemically
Infused Endothelial Progenitor Cells
[0086] To test the hypothesis that shock wave-induced up-regulation
of chemoattractant factors such as VEGF might indeed enhance the
recruitment of systemically injected human EPCs, EPCs were labeled
with a red fluorescent cell tracker and infused intravenously 24 h
after shock wave therapy of the right hind limb. Since the best
results for VEGF staining in cyrosections had been obtained using
energy of 0.43 mJ/mm.sup.2, the following experiments were
performed using the same flux density.
[0087] Animal model. Twenty-four hours after shock wave treatment
of the right hind limb, CM-Dil.sup.+ (red fluorescent) human EPCs
(1.times.10.sup.6) were intravenously injected. The animals were
sacrificed after 72 h and the tissue was evaluated for red
fluorescent EPCs incorporated into vessel structures. The number of
red fluorescent cells in the shock wave-treated right versus the
untreated left hind limb was analysed.
[0088] Clear evidence was found for homing of the injected human
EPCs to sites of shock wave treatment (right hind limb, FIG. 3a).
EPCs were found incorporated into vessels structures (FIG. 3a,
upper left panel, dotted line). A higher magnification is given in
FIG. 3a, lower left panel.
[0089] Quantification of the incorporated EPC indicated that a
markedly and significantly higher number of EPCs were incorporated
in the shock wave-treated vasculature as compared to the untreated
tissue (FIG. 3b). Thus, these data provide proof-of-concept for
shock wave-induced attraction of infused ex-vivo cultured stem and
progenitor cells.
[0090] In patients with chronic tissue damage such as previous
myocardial infarction, the recruitment of stem/progenitor cells is
markedly reduced due to the low expression of chemoattractant
factors in the target tissue. Therefore, to provide evidence for
the functional relevance of the shock wave-facilitated recruitment
of EPCs, a rat model of chronic hind limb ischemia was used.
[0091] Hind limb ischemia model. The in vivo neovascularization
capacity of infused human EPC was investigated in a rat model of
hind limb ischemia, by use of 5 wk old athymic nude rats (Charles
River Laboratory) weighing 100-120 g. The proximal portion of the
femoral artery including the superficial and the deep branch as
well as the distal portion of the saphenous artery were occluded
using an electrical coagulator. The overlying skin was closed using
surgical staples. Three weeks after induction of hind limb
ischemia, chronic ischemia was assessed by Laser Doppler imaging.
Only rats with evidence for chronic ischemia were randomized for
one of the four treatment groups: TABLE-US-00001 Shock wave Group
pretreatment EPC infusion 1 - - 2 - + 3 + - 4 + +
[0092] EPCs were infused 24 hours after shock wave
pretreatment.
[0093] Limb perfusion measurements. After two weeks, the ischemic
(right)/non-ischemic (left) limb blood flow ratio was determined
using a laser Doppler blood flow imager (Laser Doppler Perfusion
Imager System, moorLDI.TM.-Mark 2, Moor Instruments, Wilmington,
Del.). Before initiating scanning, mice were placed on a heating
pad at 37.degree. C. to minimize variations in temperature. After
twice recording laser Doppler color images, the average perfusions
of the ischemic and non-ischemic limb were calculated. To minimize
variables including ambient light and temperature, calculated
perfusion is expressed as the ratio of ischemic to non-ischemic
hind limb perfusion.
[0094] The data indicate that either EPC injection alone or shock
wave pretreatment alone significantly enhance limb perfusion as
compared to untreated control animals (FIGS. 4A and 4B). However,
limb perfusion was further enhanced by the combined treatment of
the animals. These data provide evidence for the functional impact
of shock wave-facilitated recruitment of EPC.
* * * * *