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There is strong evidence behind the synergetic relation- rheumatoid arthritis and lichen planus. However, they
ship between angiogenesis and chronic inflammation in are not curative and have significant limitations (9–11).
pathological conditions. The two phenomena share a Oral lichen planus (OLP) is a mucocutaneous,
cross-talk in many chronic inflammatory conditions chronic inflammatory disease affecting 1–2% of the
with separate etiopathogenic origins, including psoria- adult population (12) and is sub-classified into reticular,
sis, rheumatoid arthritis, Crohn’s disease, diabetes, and atrophic, and erosive forms. In the erosive form, the
lichen planus (1–3). In chronic inflammations, inflam- surface epithelium desquamates and the areas of ulcera-
matory cells secrete growth factors, proteases, and tion and erosion persist (13). Vascular endothelial
cytokines that stimulate extracellular matrix degrada- growth factor (VEGF) is believed to be the principal
tion, endothelial cell growth, and migration, thus pro- regulatory component behind endothelial cell prolifera-
moting angiogenesis. Conversely, blood vessels boost tion and differentiation involved in the pathogenesis
chronic inflammation providing cytokines, oxygen, and and progression of OLP (14, 15). As a result, it seems
nutrients, enabling inflammatory-cell chemotaxis (4–7). likely that suppression of new blood vessel formation
This current evolving data led to continuous pursuit of would delay the progression of the disease. Preventing
targeted therapy against both angiogenesis and chronic angiogenesis would minimize extravasation of immune
inflammation. Targeting angiogenesis can dramatically cells and abrogate pro-inflammatory cytokines (16, 17).
decrease the inflow of immune cells, reducing produc- Thus, pairing the positive VEGF immunoreaction in
tion of inflammatory and proteolytic moderators and OLP and the occasional unfavorable response to rou-
thus preventing nutrient supply to an active inflamma- tine corticosteroid drugs, angiogensisis can be an excel-
tory process (8). lent target for such new therapeutic modalities. This is
Currently, non-steroidal anti-inflammatory drugs backed up by several reports stating that drugs target-
(NSAIDs) and corticosteroids are the mainstay for ing VEGF can reverse the severity and progression of
treating most chronic inflammatory disorders, including chronic inflammatory diseases through inhibiting
120 Mahmoud and Afifi
VEGF (18, 19). Several preclinical studies have MA, USA), three times a day for 3 wk. Patients were asked
reported a targeted therapeutic approach against angio- to apply the topical treatment after proper mouth hygiene
genesis in chronic inflammatory disorders. Thus, non- (to make sure no food debris surrounding the lesions) using
specific anti-angiogenic compounds, such as TNP-470, a pad of washed fingers or the applicators. The lesion should
taxol, and thalidomide, have been shown to inhibit preferably have been dried first for effective adherence.
Patients were also advised to avoid food for a period of time
neovascularization and pannus formation in animal after treatment application.
models of arthritis (20–22). Moreover, other studies
have stated that cyclooxygenase-2 (COX-2)-mediated
drugs down-regulate VEGF-induced angiogenesis in Clinical follow up
psoriaris (23). Bevacizumab is a humanized monoclonal Patients were evaluated in seven follow-up sessions: after
antibody that competitively inhibits the VEGF-A fam- 1 wk; and then every 2 wk for a period of 3 months. The
ily in the extracellular space. It is a well-recognized following parameters were assessed at every follow-up
treatment for ocular neo-vascularization (ONV) and visit.
cancer (24–27). As VEGF seems to play a central role (i) Pain intensity, using the visual analog scale from 0 to
in the progression of pathological ONV and OLP (8, 10 cm (31).
27), it seems logical to investigate the therapeutic effi- (ii) Lesion size, which was measured using calibrated Wil-
cacy of intralesional injection of anti-VEGF (beva- liams periodontal probes and scored according to previ-
cizumab) in the management of erosive OLP. ously described criteria (score 5: white striae with erosive
Therefore, the aim of the study was to investigate the area >1 cm2; score 4: white striae with erosive area
effect of bevacizumab injections on atrophic/erosive <1 cm2; score 3: white striae with atrophic area >1 cm2;
OLP over a 3-month period and compare the results score 2: white striae with atrophic area <1 cm2; and score
with those of standard treatment. 0: no lesions). The erosive/atrophic area was calculated by
multiplying the maximum diameter (mm) of each erosive
lesion by the maximum width.
(iii) Improvement of each lesion (efficacy indices, EI),
Material and methods which was measured as follows: no improvement
(EI = 0); mild improvement (0 < EI < 25%); moderate
The current study followed the guidelines of the Helsinki improvement (25% ≤ EI < 75%); marked improvement
Declaration, and it was approved by the Institution Ethics (75% ≤ EI < 100%); and healed (EI = 100%) (32, 33).
Committee (Faculty of Dentistry, Alexandria University,
Egypt) for the use of human subjects in research.
Informed consent was obtained from each participant. Blinding
One clinician carried out the delivery of treatment for the
Patients and study design study and control groups, whereas a different clinician,
who was blind to the treatments, performed pre- and post-
The study included 40 patients diagnosed with atrophic/ therapeutic, and follow-up evaluations.
erosive OLP lesions in the buccal mucosa, based on a pre-
viously established inclusion criteria and clinical classifica-
tion (28). All participants were experiencing their first Histopathology
onset of lesions and did not have involvement of the skin,
vulvae, or other anatomic sites. Patients were diagnosed Tissue incisional biopsies were collected from areas most
based on clinical and histopathologic features according to representative of the erosive OLP lesions. This was per-
the World Health Organization (WHO) criteria (29). The formed before and 1 wk after treatment to confirm clinical
lesions were ≤4 cm2 in size, similar bilateral forms had <1 diagnosis and for further purposes.
cm difference in size. Specimens were fixed in 10% neutral buffered formalin,
The exclusion criteria were: (i) any uncontrolled sys- processed, and embedded in paraffin wax. Serial sections
temic disease; (ii) the presence of a lichenoid reaction of 4 lm thickness were cut, placed on glass slides, and
caused either by drugs or amalgam restorations (and con- stained using hematoxylin and eosin for routine
firmed histologically); (iii) the use of corticosteroid for at histopathologic examination using light microscopy.
least 3 months before the study; (iv) pregnant or lactating
women, or women taking contraceptive pills; and (v) other Immunohistochemistry
types of OLP. Demographics and baseline clinical findings,
including symptoms and sites of the lesions, were Immunohistochemical staining was performed using strep-
recorded. tavidin–biotin (17). Immunoexpression of VEGF was used
Of the 40 participants, 20 were chosen randomly to be to compare the angiogenesis activity rates before, and
injected with intralesional bevacizumab (test group). Beva- 1 wk after, bevacizumab treatment (34). In addition, inter-
cizumab (100 mg/4 ml; Roche, New York, NY, USA) was leukin-8 (IL-8) immunoexpression, which is directly related
diluted with distilled water to 2.5 mg/0.5 ml (27). Intra- to the course of OLP development, was assessed (28).
lesion injections were administered into the connective tissue Anti-IL-8 , anti-VEGF antibodies were used to evaluate
below the atrophic/erosive lesions (i.e. into adjacent unaf- the efficacy of the introduced treatment in comparison
fected mucosa) (30). After each injection, the participants with the control side. Briefly, tissue sections were deparaf-
were observed for 30 min for any sign of acute hypersensi- finized, rehydrated, and treated with 3% H2O2 to quench
tivity reactions or other adverse events. The remaining 20 endogenous peroxidase activity. After washing in 10 ml of
patients (control group) were instructed to use triamci- Tris-buffered saline (TBS), sections were incubated with
nolone acetonide in orabase (Bristol-Myers Squibb, Devens, 10% normal goat serum (Zymed Laboratories, San
Bevacizumab to treat oral lichen planus 121
Table 1
Clinical signs and symptoms associated with patients with oral lichen planus (OLP) at inclusion
Study group (n = 20) 43 1.3 100 6.90 0.4 13.3 0.88 0.72 0.02
Control group 42 1.1 100 6.79 0.4 13.5 1.02 0.75 0.04
(n = 20)
A B
C D
A B
C D
A B C D
E F G H
Fig. 7. Transmission electron microscopy (TEM) micrographs of oral lichen planus (OLP) tissue sections, before and 1 wk after
injection of bevacizumab. (A) Epithelial cells show marked separation and break down of their desmosomal junctions (red aster-
isk). Marked sub-epithelial bacterial invasion can be seen (red arrow) (91000). (B) Part of the oral epithelium adjacent to lamina
propria. Basal cells undergoing apoptotic changes are indicated (red arrow). Note the discontinuity in the basement membrane
(white arrow) (94000). (C) The epithelial cells show marked, wide intercellular spaces (red asterisk). Lymphocytes are seen among
the separated disorganized epithelial cells (arrow) (92000). (D) Apoptotic basal cells are in close proximity to red blood cells
(RBC) (92000). (E) Well-organized prickle cells of the oral epithelium are evident, with normal cytoplasmic electron density, nor-
mal nuclear appearance, and normal density of the desmosomal junctions with the adjacent cells (9500). (F) Formation of base-
ment membrane (arrow) (94000). (G) Two well-organized prickle cells showing normal nuclear density and normal desmosomal
attachments (92000). (H) Well-organized prickle cell of the oral epithelium, with normal cytoplasmic electron density, normal
nuclear appearance, and normal density of the desmosomal junctions with the contacting cells (92500).
Bevacizumab to treat oral lichen planus 125
bevacizumab can now be considered as the treatment and that targeting VEGF could rapidly terminate the dis-
of choice for chronic inflammatory angiogenesis-depen- ease process. Intralesional bevacizumab injections seem
dent diseases, including atrophic/erosive OLP. to be a relatively safe, effective, and well-tolerated option
Ultrastructural examinations of the untreated for the treatment of progressive OLP. Intralesional beva-
atrophic/erosive OLP biopsies revealed morphologic cizumab therapy may be a favorable alternative in con-
features that were similar to previously reported ultra- traindicated, non-responsive, or resistant patients.
structural features associated with OLP (39). Basal lam- Future controlled prospective randomized trials are nec-
ina exhibited breaks, and degenerative changes in the essary to learn more about the long-term safety, tolera-
basal cell layer were evident. The prickle cell layer bility, and efficacy of bevacizumab. Additional research
showed marked widening of the intercellular spaces and will be needed, including a larger sample size, an
loss of desmosomal attachments. Apoptotic changes extended study period, and a panel of different beva-
were seen, including increased nuclear chromatin, cyto- cizumab doses, to validate these results.
plasmic vacuolization, and blebbing. As a result of the
Acknowledgements – This study was financially supported by
breaks in the basement membrane, lymphocytes were
Alexandria University, Egypt.
trapped within the basal and prickle cell layers. There
was a marked bacterial infection adherent to the disor-
Conflicts of interest – The authors declare no conflicts of interest.
ganized layer of epithelial cells. This is likely to be seen
due to the superimposed inflammation. Both the breaks
in the basement membrane and sub-epithelial bacterial
invasion can explain the increased pain intensity associ- References
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124 Mahmoud and Afifi
A B C D
E F G H
Fig. 7. Transmission electron microscopy (TEM) micrographs of oral lichen planus (OLP) tissue sections, before and 1 wk after
injection of bevacizumab. (A) Epithelial cells show marked separation and break down of their desmosomal junctions (red aster-
isk). Marked sub-epithelial bacterial invasion can be seen (red arrow) (91000). (B) Part of the oral epithelium adjacent to lamina
propria. Basal cells undergoing apoptotic changes are indicated (red arrow). Note the discontinuity in the basement membrane
(white arrow) (94000). (C) The epithelial cells show marked, wide intercellular spaces (red asterisk). Lymphocytes are seen among
the separated disorganized epithelial cells (arrow) (92000). (D) Apoptotic basal cells are in close proximity to red blood cells
(RBC) (92000). (E) Well-organized prickle cells of the oral epithelium are evident, with normal cytoplasmic electron density, nor-
mal nuclear appearance, and normal density of the desmosomal junctions with the adjacent cells (9500). (F) Formation of base-
ment membrane (arrow) (94000). (G) Two well-organized prickle cells showing normal nuclear density and normal desmosomal
attachments (92000). (H) Well-organized prickle cell of the oral epithelium, with normal cytoplasmic electron density, normal
nuclear appearance, and normal density of the desmosomal junctions with the contacting cells (92500).