Angiogenin (ANG) was originally identified as a tumour angiogenic factor, but its biological activity has been extended from inducing angiogenesis to stimulating cell proliferation and promoting cell survival through stimulating ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation 1.
In cases of mycosis fungoides (MF), angiogenesis proceeds in step with clinical progression. New vessels favor invasion and metastasis, which could explain loss of epidermotropism and invasion of lymph nodes and parenchymal organs observed in aggressive stages of MF 2. Patients with hematological malignancies show increased serum ANG levels, which are related with poor overall survival 3.
It has been also shown that ANG inhibits polymorphonuclear leucocyte degranulation 4 and thus displays immunosuppressive activity 5. Regulatory cytokines such as interleukin (IL)-6, a major inducer of acute-phase proteins, stimulate the synthesis and secretion of ANG mRNA and also increase the amount of ANG protein 6.
Although angiogenesis may not be the primary event in the pathogenesis of psoriasis, understanding the pathways leading to angioproliferation may help in finding novel antipsoriatic drugs.
The aim of the present study was to estimate the tissue level of ANG in both plaque-stage MF and to compare it to the level in cases with chronic plaque psoriasis.
Patients and methods
This comparative study was conducted on 50 participants selected from the Dermatology Outpatient Clinic, Kasr El Aini Hospital, after receiving the approval of the Dermatology Research Ethics (Derma-REC) Committee. Informed written consents were retrieved from all participants.
Twenty patients with plaque-stage MF, 20 patients with chronic plaque psoriasis, and 10 age-matched and sex-matched healthy volunteers were enrolled in this study. The diagnosis was made on clinical bases and confirmed by skin biopsies. All patients received no topical or systemic therapy at least 4 weeks before taking skin biopsies.
Patients were subjected to thorough history taking, physical and dermatological examination to determine the distribution, clinical variant, and extent of lesions. Routine laboratory investigations (in the form of complete blood count with differential, liver function tests, kidney function tests, and blood sugar), lymph node assessment, chest radiography, and abdominal ultrasound were done for proper staging of the disease.
Staging was based on revised criteria proposed by the International Society for Cutaneous Lymphomas and the cutaneous lymphoma task force of the European Organization for Research and Treatment of Cancer 7.
Psoriasis area and severity index (PASI) score was calculated for each psoriasis patient. Schmitt and Wozel 8 in chronic plaque-type psoriasis and PASI in more than 12 severe chronic plaque-type psoriasis.
- Patients not taking any medications in the last 4 weeks.
- Both sexes were included.
- Below 18 years of age.
- Patients with any other type of psoriasis or MF other than the plaque type.
- Patients suffering from any systemic illness (liver or kidney diseases, autoimmune diseases, or diabetics).
- Patients with associated skin diseases.
One skin biopsy was taken from each patient and control. The selected area was cleaned with alcohol and a local anaesthetic (xylocaine 2%) was injected subcutaneously. Then a 6-mm punch biopsy was obtained. The skin biopsy was stored as a frozen section at −80°C. All specimens were sent to the Biochemistry Department, Faculty of Medicine, Cairo University, for detection of the ANG level by PCR.
Detection of angiogenin gene expression by real-time-PCR
Reagents for RNA extraction and cDNA synthesis:
- SV total RNA extraction kit supplied by Promega (Madison, Wisconsin, USA).
- DEPC-treated water.
- SuperScript II reverse transcriptase and buffer, oligo (dT) 12–18, or random hexamer primer; dNTP.
- RNase inhibitor.
Reagents for qPCR:
- SYBR Green (Thermo Fisher Scientific, Waltham, Massachusetts, USA) I qPCR mixture (this mixture includes dNTP, Taq DNA polymerase, reaction buffer, and the fluorescent dyes gene specific primers).
- Plastic: white strip qPCR tube (200 µl volume) with optical clear strip caps or white 96-well qPCR plates with optical clear seal sheets and press applicator.
- qPCR equipment, step one Applied Biosystem (Qiagene, Hilden, Germany).
- Housekeeping gene: β-actin.
- Extraction of RNA from the skin tissue:
- Total RNA was extracted from the skin tissue using SV Total RNA Isolation system (Promega).
- Homogenization of the skin tissue.
- RNA purification.
- Determination of RNA yield and quality.
- Reverse transcription into cDNA:
- The extracted RNA was reverse transcribed into cDNA using the RT-PCR kit (Stratagene, La Jolla, California, USA).
- Procedure: 3 μl of random primers were added to the 10 μl of RNA, which was denatured for 5 min at 65°C in the thermal cycler.
- The RNA primer mixture was cooled to 4°C.
- The cDNA master mix was prepared.
- Total volume of the master mix was 19 μl for each sample. This was added to the 13 μl RNA-primer mixture resulting in 32 μl of cDNA.
- The last mixture was incubated in the programmed thermal cycler for 1 h at 37°C, followed by inactivation of enzymes at 95°C for 10 min, and finally cooled at 4°C. Then RNA was changed into cDNA.
- Quantitative real-time PCR:
- The gene-specific forward and reverse primer pairs were normalized. Each primer (forward and reverse) concentration in the mixture was 5 pmol/µl (Table 1).
- The experiment and the following PCR program was set up:
- 50°C 2 min, one cycle.
- 95°C 10 min, one cycle.
- 95°C 15 s, 60°C 30 s, 72°C 30 s; 40 cycles.
- 72°C 10 min, one cycle.
- A real-time-PCR mixture of 50 µl.
- The following mixture was prepared in each optical tube:
- 25 µl SYBR Green Mix (2×).
- 0.5 µl kidney cDNA.
- 2 µl primer pair mix (5 pmol/µl each primer).
- 22.5 µl H2O.
- After PCR is finished, the tubes from the machine were removed.
- The real-time-PCR result was analysed with the step one Applied Biosystem software.
At the end of a qPCR running with SYBR Green chemistry, the relative quantification was used.
Data analysis and relative quantitation
At the end of a qPCR running with SYBR Green chemistry, the relative quantification was used according to the Applied Biosystem software (La Jolla, California, USA) 9.
Statistical package for the social sciences (SPSS) program version 17 (SPSS Inc., Chicago, Illinois, USA) was used for the analysis of data. Data were summarized as median, mean±SD, number, and percentage. The t-test was used for the analysis of quantitative data when normally distributed. One-way analysis of variance test was used for the analysis of more than two quantitative variables and the Mann–Whitney U-test for the quantitative not normally distributed data. Pearson’s correlation was done to test the linear relation between quantitative variables. P value is considered significant if less than 0.05.
This comparative study included 20 patients with chronic plaque psoriasis (group A), 20 patients with plaque stage MF (group B), and 10 age-matched and sex-matched healthy volunteers serving as controls (group C).
Group A: Their ages ranged from 20 to 52 years with a mean±SD of 37.6±10.5 years. They were 13 (65%) men and seven (35%) women. The duration of the disease ranged from 1 to 35 years with a mean±SD of 11.8±9 years. The extent of body involvement ranged from 9 to 70% with a mean of 30.2±17.4%. The course of disease was progressive in seven (35%) patients and in remissions and exacerbations in 13 (65%) patients. The PASI score ranged from 4.8 to 54 with a median of 14.1.
Group B: Their ages ranged from 18 to 70 years with a mean±SD of 35.9±14.8 years. They were 13 (65%) men and seven (35%) women. The duration of the disease ranged from 1 to 25 years with a mean±SD of 6.5±6 years. The extent of body involvement ranged from 10 to 50% with a mean±SD of 20.5±12.3%. The course of disease was progressive in 17 (85%) patients and in relapse in three (15%) patients. Nine (45%) patients were in stage IA of the disease and 11 (55%) patients were in stage IB.
Group C: Their ages ranged from 21 to 56 years with a mean±SD of 34.2±9.8 years. They were seven (70%) men and three (30%) women.
Group A: The ANG levels ranged from 0.08 to 0.45 with a mean±SD of 0.17±0.104 µg/ml. A significant negative correlation was detected between the age of the patients and the ANG levels (r=−0.459, P=0.012) (Fig. 1). On the other hand, no correlations were detected between ANG levels and duration, extent of the disease, or PASI score (P=0.740, 0.680, 0.391, respectively).
No statistically significant difference was detected in ANG level between women and men (P=0.781).
Group B: The ANG levels ranged from 2.7 to 10.5 with a mean±SD of 7.66±2.349 µg/ml. A significant positive correlation was detected between ANG levels and the extent of lesions (r=0.517, P=0.019) (Fig. 2), while no correlations were detected between ANG levels and age of the patients or the duration of disease (P=0.329, 0.098, respectively).
No statistically significant difference was detected in the ANG level between women and men in group B (P=1.000).
In patients with stage 1 A, ANG levels ranged from 2.7 to 9 with a median of 5.1 µg/ml, while in patients with stage 1B, ANG levels ranged from 5 to 10.5 with a median of 9.3 µg/ml. A high statistically significant difference was detected between both groups as regards the ANG level (P=0.003) (Fig. 3).
Group C: included seven (70%) men and three (30%) women with a mean±S.D age of 34.2±9.8 years. Their skin biopsies showed that the ANG levels range from 1.07 to 4.02 with a mean±SD of 1.85±0.864 µg/ml.
Comparing the angiogenin level in the studied groups
Comparing the ANG level in group A versus group C showed that the ANG level was statistically lower in group A than in group C with a median of 0.14, 1.7 µg/ml, respectively (P<0.001).
Comparing the ANG level in group B versus group C showed that the ANG level was statistically higher in group B than in group C with a median of 8.3, 1.7 µg/ml, respectively (P<0.001).
Comparing the ANG level in group A versus group B showed that the ANG level was statistically higher in group B than in group A with a median of 8.3, 0.14 µg/ml, respectively (P<0.001) (Fig. 4).
This study was primarily based on the idea of the pathological angiogenic process which occurs in chronic inflammation, where angiogenesis is needed for disease development 10 and its correlation with tumour growth (S-phase fraction) in some lymphoproliferative diseases of T-cell lineages 11. Thus, psoriasis and MF patients were enrolled in the current study which aimed at the estimation of the tissue level of ANG in psoriatic, MF patients, and controls by the PCR technique.
The present study has shown significantly lower ANG level in psoriatic patients than the controls, which was an unexpected finding, where psoriasis has been studied since 1972 in the light of its being angiogenic in nature. However, this could be explained by the fact that angiogenesis in psoriasis is under the control of several angiogenic factors, where ANG might not have a major role. Proangiogenic mediators, such as tumour necrosis factor, vascular endothelial growth factor (VEGF), hypoxia-inducible factor, IL-8, or angiopoietins, are enriched in psoriatic skin 12. A proangiogenic role has also been attributed to the Th17 cytokine IL-17 13,14. As transforming growth factor-α induces VEGF expression and secretion by epidermal keratinocytes in vitro 15 and is overexpressed in suprabasal keratinocytes of psoriatic skin, transforming growth factor-α might be responsible for the epidermal VEGF upregulation during psoriasis 16.
In accordance with our results, Miyagaki et al. 3, who measured the serum and tissue levels of ANG in psoriasis patients, documented significantly lower levels of ANG in psoriasis patients than the normal values. In addition, Wang et al. 17 reported that the basic fibroblast growth factor which is increased in psoriatic skin 18 negatively regulates ANG expression.
ANG was known as an inhibitor of neutrophil degranulation since 1994. Most plasma-derived molecules released during an inflammatory response, for example, endotoxins, ILs, etc. are known to stimulate PMNL functions rather than inhibit them. Therefore, at first glance circulating ANG is a compound to counterbalance these stimulating agents 4. Another study carried out on uremic patients concluded that high ANG plasma levels protect against lactoferrin release from PMNL during extracorporeal circulation in chronic uremic patients. A decrease of plasma ANG of between 20 and 40% during extracorporeal circulation, however, results in marked PMNL lactoferrin release 19.
Miyagaki et al. 3 suggested that decreased ANG expression in the psoriatic skin might allow neutrophil degranulation in lesional skin, activating downstream inflammatory processes and causing psoriasis progression. They hypothesized that the increase in ANG induced by infliximab may help prevent neutrophil activation.
The current study also showed a negative correlation detected between the ANG levels and the age of psoriatic patients. This correlation was neither detected in MF group nor the controls. This may be attributed to the studies that confirmed that psoriasis is linked to several cardiovascular risks. Eder and Gladman 20 stated that population-based studies have found an increased cardiovascular risk in patients with psoriasis and higher risk is found with severe disease phenotypes. Rivard et al. 21 concluded that angiogenesis was impaired as a function of age. Although this study was performed on animal models with hind-limb ischaemia, it confirmed the significant reduced capability for collateral vessel development in response to ischemia in older models. The number of blood vessels that were angiographically visible and the number of capillaries per unit area identified histologically were both significantly reduced in old versus young animals. The latter finding is consistent with the observation of Tomanek et al. 22 who showed that myocardial angiogenesis related to left ventricular hypertrophy is attenuated in an age-dependent manner, another study done on animal models.
On the other hand, no significant correlations were detected between ANG level and duration, extent of the disease, or PASI score. This was in agreement with the study of Miyagaki et al. 3 who reported absence of correlation between serum ANG level and PASI score.
As for MF patients, according to the current results, the ANG level was significantly higher than its level in the control skin. There was also a significant increase in the ANG level in MF stage IB than in stage IA which was in agreement with Mazur et al. 23, who stated that biopsies obtained from the lesional skin of patients with CTCL exhibited stage-dependent increase in angiogenesis.
Moreover, a significant positive correlation was detected between ANG level and the extent of lesions (r=0.517, P=0.019), while there were no correlations detected between ANG level and age of the patients or the duration of disease.
This is in accordance with the results detected by Miyagaki et al. 3, who estimated the ANG level in serum and lesional skin of CTCL. Serum ANG levels in patients with CTCL were significantly higher than those in healthy controls. When classified with the types of skin lesions (patch, plaque, and tumour), serum and mRNA expression of ANG in lesional skin were elevated only in erythrodermic CTCL patients. Immunohistochemical study has shown that ANG was expressed by keratinocytes, endothelial cells, and infiltrating lymphocytes in CTCL. Their results suggest that enhanced ANG expression may be related with a poor prognosis of erythrodermic CTCL. As ANG acts as an inhibitor of polymorphonuclear leucocyte degranulation, ANG may also be linked to impaired host defense in erythrodermic CTCL.
Interestingly, erythrodermic CTCL patients, who show elevated levels of ANG in the skin and sera, are frequently susceptible to the infection by bacterial, viral, and fungal pathogens 24. Opposing this data, a study was done by Hooper et al. 25 who demonstrated that in addition to the role of ANG in tumour-associated angiogenesis, circulating ANG induced during inflammation exhibits microbicidal activity against systemic bacterial and fungal pathogens, suggesting that they contribute to systemic responses to infection. These results establish ANG as a family of endogenous antimicrobial proteins.
Comparing the ANG level in MF group with the psoriasis group in our study showed a highly significant increase in the ANG level in the MF group than its level in psoriasis patients. This might be explained by the assumption that the neovascularization sufficient for tumour progression and spread is likely to be more than that involved in chronic inflammatory disease such as psoriasis.
This study highlighted the possible prognostic effect of ANG in different stages of MF; however, it did not prove any role in psoriasis. Further studies are still needed to verify such findings in larger patient groups and different clinical types of psoriasis and MF patients. As angiogenesis is becoming a key phenomenon in the development of psoriasis and MF, it remains open to what extent the anti-angiogenic properties of the treatments can prove their efficacy in both disease groups.
No financial support.
Conflicts of interest
There are no conflicts of interest.
1. Li S, Hu GF. Emerging role of angiogenin
in stress response and cell survival under adverse conditions. J Cell Physiol 2012; 227:2822–2826.
2. Willemze R, Beljaards RC, Meijer CJ. Classification of primary cutaneous T-cell lymphomas. Histopathology 1994; 24:405–415.
3. Miyagaki T, Sugaya M, Suga H. Angiogenin
levels are increased in lesional skin and sera in patients with erythrodermic cutaneous T cell lymphoma. Arch Dermatol Res 2012; 304:401–406.
4. Tschesche H, Kopp C, Hörl WH, Hempelmann U. Inhibition of degranulation of polymorphonuclear leukocytes by angiogenin
and its tryptic fragment. J Biol Chem 1994; 269:30274–30280.
5. Matousek J, Soucek J, Riha J, Zankel TR, Benner SA. Immunosuppressive activity of angiogenin
in comparison with bovine seminal ribonuclease and pancreatic ribonuclease. Comp Biochem Physiol 1995; 112B:235–241.
6. Verselis SJ, Olson KA, Fett JW. Regulation of angiogenin
expression in human HepG2 hepatoma cells by mediators of the acute-phase response. Biochem Biophys Res Commun 1999; 259:178–184.
7. Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler R, et al. Revisions to the staging and classification of mycosis fungoides
and Sezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 2007; 110:1713.
8. Schmitt J, Wozel G. The psoriasis
area and severity index is the adequate criterion to define severity in chronic plaque-type psoriasis
. Dermatology 2005; 210:194–199.
9. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45.
10. Wieder T, Braumüller H, Kneilling M, Pichler B, Röcken M. T cell-mediated help against tumors. Cell Cycle 2008. 2974–2977.
11. Kittas C, Hansmann ML, Borisch B, Feller AC, Lennert K. The blood microvasculature in T-cell lymphomas. A morphological, ultrastructural and immunohistochemical study. Virchows Arch A Pathol Anat Histopathol 1985; 405:439–452.
12. Creamer D, Sullivan D, Bicknell R, Barker J. Angiogenesis in psoriasis
. Angiogenesis 2002; 5:231–236.
13. Starnes T, Robertson MJ, Sledge G, Kelich S, Nakshatri H, Broxmeyer HE, Hromas R. Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis and endothelial cell cytokine production. J Immunol 2001; 167:4137–4140.
14. Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, et al. Interleukin-17 promotes angiogenesis and tumor growth. Blood 2003; 101:2620–2627.
15. Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, et al. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis
. J Exp Med 1994; 180:1141–1146.
16. Heidenreich R, Röcken M, Ghoreschi K. Angiogenesis drives psoriasis
pathogenesis. Int J Exp Pathol 2009; 90:232–248.
17. Wang J, Yang J, Yuan D, Wang J, Zhao J, Wang L. Effects of basic fibroblast growth factor on angiogenin
expression and cell proliferation in H7402 human hepatoma cells. J Genet Genomics 2009; 36:399–407.
18. Freeman MR, Schneck FX, Gagnon ML. Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis. Cancer Res 1995; 55:4140–4145.
19. Schmaldienst S, Oberpichler A, Tschesche H, Hörl WH. Angiogenin
: a novel inhibitor of neutrophil lactoferrin release during extracorporeal circulation. Kidney Blood Press Res 2003; 26:107–112.
20. Eder L, Gladman DD. Atherosclerosis in psoriatic disease: latest evidence and clinical implications. Ther Adv Musculoskelet Dis 2015; 7:187–195.
21. Rivard A, Fabre JE, Silver M, Chen D, Murohara T, Kearney M, et al. Age-dependent impairment of angiogenesis. Circulation 1999; 99:111–120.
22. Tomanek RJ, Aydelotte MR, Torry RJ. Remodeling of coronary vessels during aging in purebred beagles. Circ Res 1991; 69:1068–1074.
23. Mazur G, Woźniak Z, Wróbel T, Maj J, Kuliczkowski K. Increased angiogenesis in cutaneous T-cell lymphomas. Pathol Oncol Res 2004; 10:34–36.
24. Axelrod PI, Lorber B, Vonderheid EC. Infections complicating mycosis fungoides
and Sézary syndrome. JAMA 1992; 267:1354–1358.
25. Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat Immunol 2003; 4:269–273.