Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT05112744 |
| Other study ID # |
APHP210749 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
November 2021 |
| Est. completion date |
November 2022 |
Study information
| Verified date |
October 2021 |
| Source |
Assistance Publique - Hôpitaux de Paris |
| Contact |
Hester Colboc, MD |
| Phone |
00 33 6 03 61 16 23 |
| Email |
hester.colboc[@]aphp.fr |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational [Patient Registry]
|
Clinical Trial Summary
The spectrum of pathologies accompanied by tissue mineral deposits is wide. In dermatology,
several pathologies are associated with calcium mineral deposits, such as calciphylaxis and
pseudoxanthoma elasticum (PXE). However, few studies have been carried out on the chemical
characteristics of these deposits, their implication on the pathophysiology and their
consequences. This motivated our two previous studies on the characterization of skin mineral
deposits during calciphylaxis and sarcoidosis. We have shown that these deposits were most
often composed of carbapatite and preferentially localized to elastic fibers. Most calcifying
dermatoses are preceded by an inflammatory skin condition. Some authors suspect the digestion
of elastin by metalloproteinase (MMP) of the extracellular matrix, thus creating nucleation
nuclei favoring phosphocalcic deposits. We thus wish to study the structural alteration of
dermal elastic fibers during calcifying dermatoses using multiphoton microscopy, a tool
available at the Laboratoire d'Optique et Biosciences (LOB) at the Ecole Polytechnique.
Multiphoton microscopy presents several contrast modes that can be used in parallel and
without marking. This makes it possible to identify constituent elements of tissues without
the use of artificially added fluorescent dyes or proteins, for example fibrillar collagen by
the so-called "SHG" contrast and elastin by its intrinsic fluorescence. It is then possible
to deeply image an intact tissue, without staining, by specifically visualizing its various
components. Used in several studies on the skin, including the LOB, multiphoton microscopy
has shown its interest in the characterization of dermal fibers, in particular elastin and
collagen fibers, but also in the structural study of these and of their possible alteration.
It has thus been applied to the study of skin aging, but also of pathologies leading to
degeneration of elastic fibers (PXE) or collagen (Marfan syndrome). The main objective of our
project is to characterize the structural alterations of elastic fibers during calcifying
dermatoses. The secondary objectives are to study the consequences of skin inflammatory
phenomena on the deterioration of elastic fibers and to identify a possible nucleus of
phospho-calcium deposits within elastic dermal and vascular fibers. We will thus study human
skin biopsies already carried out in the context of the diagnosis of these calcifying
dermatoses, skin biopsies from the murine model of PXE and in control, human biopsies of
healthy skin from patients of different ages (excision margin of skin tumors). This project
should provide a better understanding of the genesis of skin phosphocalcic deposits and
provide therapeutic avenues for treating them and limiting their occurrence.
Description:
The variety of pathologies accompanied by mineral deposits in tissues is large (cancers,
infectious processes, environmental diseases) (1). In dermatology, several pathologies are
associated with calcium mineral deposits, such as calciphylaxis and pseudoxanthoma elasticum
(PXE). To date, few studies have been carried out on the chemical characteristics of
cutaneous mineral deposits, their physiopathological consequences, the occurrence of
associated pathologies and their repercussions on human organs.
This has motivated the PhD project of Hester COLBOC, M.D., on the characterization of calcium
deposits in various calcifying dermatosis, including calciphylaxis (2) and sarcoidosis (3).
This fundamental research project is carried out in the framework of a collaboration between
the LCP (Institute of Physico-Chemistry, CNRS, Paris-Saclay University) and AP-HP, in
particular in the fields of uro-nephrology (urinary lithiasis) and oncology (4)
(calcifications in breast and thyroid cancer). We have shown that skin calcifications consist
of carbapatite in calciphylaxis and calcite in sarcoidosis. We have also shown that these
deposits are mainly located in the dermal or vessel wall elastic fibres, suggesting a
molecular nucleation pathway within these fibers to be explored.
This structural and chemical description is innovative, but does not answer all the questions
that arise around these calcium deposits: why do some patients, who present a normal
phosphocalcium balance, have massive skin calcifications? Why, on the contrary, do other
patients, such as some dialysis patients, present extremely disturbed phosphocalcic balance
sheets, but no skin calcification? This paradox raises the possibility of the existence of
"nucleation foci" within the elastic dermal and vascular fibers, favouring the precipitation
of phosphocalcic deposits in some patients. Some in vitro models have explored these
nucleation foci within elastic fibres. (ref Gourgas et al) Quite remarkably, most calcifying
dermatosis are preceded by a cutaneous inflammatory state. These skin inflammatory phenomena
could also induce the precipitation of calcium deposits. Some authors thus speculate that
elastin is digested by metalloproteinase (MMP) in the extracellular matrix, thus creating
nucleation foci favouring phosphocalcic deposits. Munavalli et al. showed an increase level
of MMP in urinary sample of a patient with calciphylaxis in a context of rapid weight loss
(5). They hypothesize that elevated serum MMP levels in this patient could lead to an
alteration of the external elastic limit and vascular calcifications. This hypothesis is
supported on the other hand by other models of elastic fiber alteration, such as during skin
aging (dermatoporosis), in which there is no inflammation and no calcification.
The background hypothesis of our project is that skin inflammation leads to a specific
alteration of the elastic fibres, which is conducive to their calcification.
In order to explore this hypothesis, particularly during calciphylaxis and PXE, we propose to
use multiphoton microscopy, an imaging technique available at the Laboratoire d'Optique et
Biosciences (LOB) at the Ecole Polytechnique. Multiphoton microscopy has been developed in
the early 1990s as an alternative to confocal microscopy, to improve in-depth imaging of
biological tissues with sub-micrometric resolution. Most importantly, multiphoton microscopy
can combine several lodes of contrast in parallel and without any labeling to identify the
various elements of a tissue. Second harmonic generation (SHG) signals allow specific imaging
of unstained fibrillar collagen with unequalled sensitivity, while two-photon excited
fluorescence (2PEF) allows cellular imaging thanks to various intrinsic cellular chromophores
as well as elastin imaging in the dermis or in other tissues (see Figure 1) (6).
Multiphoton microscopy has been used in several studies on the skin, including at LOB (see
Figure 1), which have proven the high relevance of this technique for the structural
characterization of dermal fibers, particularly elastin and collagen fibers, and their
possible alteration. It has been applied to the study of skin ageing, as well as to
pathologies leading to degeneration of elastic (PXE) or collagen fibres (Marfan Syndrome)
(7-9). This technique is therefore highly appropriate for exploring the structure of elastic
fibres during cutaneous inflammatory phenomena and calcifying dermatoses.
In parallel, the cutaneous inflammatory process will be explored by other methods.
Immuno-histochemistry will be used to detect dermal MMP and X-ray fluorescence (at
Synchrotron Soleil, Diffabs line) will be used to evidence intradermal zinc and other
metallic deposits, as done in our previous studies (Figure 2).
Figure 2: X-ray fluorescence of a skin biopsy: example of correlation between dermal
inflammatory infiltrate (A) and the presence of intradermal Zinc (B), here during a lichenoid
reaction on tattoo (images recorded at the Soleil Synchrotron, Diffabs line, by S. Reguer, D.
Bazin and H. Colboc).
The main objective of our project is to characterize the structural alterations of elastic
fibers during calcifying dermatosis.
The secondary objectives are:
- to study the consequences of cutaneous inflammatory processes on the alteration of
elastic fibers,
- to identify a possible nucleation foci for the formation of phospho-calcium deposits
within the elastic dermal and vascular fibers.
Skin samples. The study will be mainly performed on human skin, but also on a mouse model of
PXE: ABCC6 KO mouse. More precisely, the material studied at LOB will be:
- Human skin biopsies already collected for the diagnosis of these calcifying dermatoses:
PXE, calciphylaxis, other calcifying inflammatory dermatoses (lupus panniculitis,
dermatomyositis), from the pathology laboratory of the Tenon hospital (Dr Moguelet).
- As controls, human biopsies of healthy skin from patients of different ages, (margin of
skin tumour removal), from the Tenon Hospital. We will focuse on patient with advanced
skin aging, especially patients with solar elastosis. This model of UV induced elastic
fibers alteration is a great control in term of non-inflammatory and non-calcified
elastic fibers damages.
- Cutaneous biopsies from the mouse model of PXE (ABCC6 KO mouse) and from matched control
mice, from INSERM Unit 1155, Tenon Hospital.
Protocols. All these biopsies will be characterized by multiphoton microscopy by combining
2PEF and SHG contrasts in order to simultaneously image elastic and collagen fibers. In a
first step, the experimental protocol will be as follows:
- Production at Tenon Hospital of several serial slides from each biopsy: 1 unstained
("white") slide for multiphoton imaging, 1 slide with usual morphological staining
(hematoxylin-eosin-safran, HES), 1 slide with staining highlighting calcium deposits
(Von Kossa) and 3 slides with immuno-histochemical labelings of the main MMPs
(antibodies against MMP-1, 2 and 9).
- imaging at LOB on a standard brightfield optical microscope (standard and polarized
transmitted light, fluorescence, DIC) of all the stained slides over a large field of
view by mosaicking; identification of regions of interest (ROI) corresponding to
inflammatory processes, tissue remodeling or mineral deposits. Note that mineral
deposits are revealed by polarized transmitted light or by phase contrast or DIC.
- X-ray fluorescence imaging at Soleil Synchrotron to identify the metallic deposits
present in the biopsies, specially Zinc. This will be done in collaboration with
Dominique Bazin (Institute of Physico-Chemistry, CNRS, Paris-Saclay University), which
has a strong expertise in mineral imaging in the Diffabs line.
- Multiphoton imaging at LOB of the identified regions of interest, or even on a large
field of view by automatically stitching several tiles. Polarization-resolved images
will also be carried out in very dense areas or in remodeled areas in order to map the
direction of collagen and elastin fibers (10).
- Automated quantitative analysis of multiphoton LOB images to measure several parameters
related to the quantity and structure of elastin. Automated quantitative analyses will
also be performed on collagen and other structures of interest observed on multiphoton
images or histological images. The results of the different imaging modalities will be
correlated on each series of slides. Note that automatic analysis is essential both to
avoid the usual biases of semi-quantitative analysis and to obtain more informative
structural parameters. For instance, the following parameters will be extracted from the
elastin images: the mean density of the elastic fibers (per cm², in the papillary dermis
and in the reticular dermis), the orientation distribution of the elastic fibers, the
entropy and the circular variance of this distribution, the degree of curvature of the
elastic fibers, their distance from the mineral deposits.
- Statistical analysis of multimodal data, based on appropriate statistical tests. A
priori non-parametric tests (Wilcoxon-Mann-Whitney) will be used in view of the
relatively small number of available human samples. A minimum of 8 patients per group
will be studied, which should be sufficient due to the accuracy and reproducibility of
the automated measurements. If several samples or image series are performed on the same
patient, nested statistical tests will be used (nested t-tests or nested Wilcoxon).
In a second step and based on the results of the first step, various items of this protocol
will be improved to access more information:
- Multiphoton imaging will be performed in intact murine skin biopsies to better highlight
the 3D organization of elastin.
- Advanced fluorescence imaging of inflammation will be performed in ex vivo murine skin,
using spectral or FLIM (fluorescence lifetime imaging microscopy) measurements
- Third Harmonic Generation (THG) will be used to visualize the structure of the mineral
deposits.
1. Bazin D, et al. Characterization and some physicochemical aspects of pathological
microcalcifications. Chem Rev. 10 oct 2012;112(10):5092-120.
The variety of pathologies accompanied by mineral deposits in tissues is large (cancers,
infectious processes, environmental diseases) (1). In dermatology, several pathologies are
associated with calcium mineral deposits, such as calciphylaxis and pseudoxanthoma elasticum
(PXE). To date, few studies have been carried out on the chemical characteristics of
cutaneous mineral deposits, their physiopathological consequences, the occurrence of
associated pathologies and their repercussions on human organs.
This has motivated the PhD project of Hester COLBOC, M.D., on the characterization of calcium
deposits in various calcifying dermatosis, including calciphylaxis (2) and sarcoidosis (3).
This fundamental research project is carried out in the framework of a collaboration between
the LCP (Institute of Physico-Chemistry, CNRS, Paris-Saclay University) and AP-HP, in
particular in the fields of uro-nephrology (urinary lithiasis) and oncology (4)
(calcifications in breast and thyroid cancer). We have shown that skin calcifications consist
of carbapatite in calciphylaxis and calcite in sarcoidosis. We have also shown that these
deposits are mainly located in the dermal or vessel wall elastic fibres, suggesting a
molecular nucleation pathway within these fibers to be explored.
This structural and chemical description is innovative, but does not answer all the questions
that arise around these calcium deposits: why do some patients, who present a normal
phosphocalcium balance, have massive skin calcifications? Why, on the contrary, do other
patients, such as some dialysis patients, present extremely disturbed phosphocalcic balance
sheets, but no skin calcification? This paradox raises the possibility of the existence of
"nucleation foci" within the elastic dermal and vascular fibers, favouring the precipitation
of phosphocalcic deposits in some patients. Some in vitro models have explored these
nucleation foci within elastic fibres. (ref Gourgas et al) Quite remarkably, most calcifying
dermatosis are preceded by a cutaneous inflammatory state. These skin inflammatory phenomena
could also induce the precipitation of calcium deposits. Some authors thus speculate that
elastin is digested by metalloproteinase (MMP) in the extracellular matrix, thus creating
nucleation foci favouring phosphocalcic deposits. Munavalli et al. showed an increase level
of MMP in urinary sample of a patient with calciphylaxis in a context of rapid weight loss
(5). They hypothesize that elevated serum MMP levels in this patient could lead to an
alteration of the external elastic limit and vascular calcifications. This hypothesis is
supported on the other hand by other models of elastic fiber alteration, such as during skin
aging (dermatoporosis), in which there is no inflammation and no calcification.
The background hypothesis of our project is that skin inflammation leads to a specific
alteration of the elastic fibres, which is conducive to their calcification.
In order to explore this hypothesis, particularly during calciphylaxis and PXE, we propose to
use multiphoton microscopy, an imaging technique available at the Laboratoire d'Optique et
Biosciences (LOB) at the Ecole Polytechnique. Multiphoton microscopy has been developed in
the early 1990s as an alternative to confocal microscopy, to improve in-depth imaging of
biological tissues with sub-micrometric resolution. Most importantly, multiphoton microscopy
can combine several lodes of contrast in parallel and without any labeling to identify the
various elements of a tissue. Second harmonic generation (SHG) signals allow specific imaging
of unstained fibrillar collagen with unequalled sensitivity, while two-photon excited
fluorescence (2PEF) allows cellular imaging thanks to various intrinsic cellular chromophores
as well as elastin imaging in the dermis or in other tissues (see Figure 1) (6).
Multiphoton microscopy has been used in several studies on the skin, including at LOB (see
Figure 1), which have proven the high relevance of this technique for the structural
characterization of dermal fibers, particularly elastin and collagen fibers, and their
possible alteration. It has been applied to the study of skin ageing, as well as to
pathologies leading to degeneration of elastic (PXE) or collagen fibres (Marfan Syndrome)
(7-9). This technique is therefore highly appropriate for exploring the structure of elastic
fibres during cutaneous inflammatory phenomena and calcifying dermatoses.
In parallel, the cutaneous inflammatory process will be explored by other methods.
Immuno-histochemistry will be used to detect dermal MMP and X-ray fluorescence (at
Synchrotron Soleil, Diffabs line) will be used to evidence intradermal zinc and other
metallic deposits, as done in our previous studies (Figure 2).
Figure 2: X-ray fluorescence of a skin biopsy: example of correlation between dermal
inflammatory infiltrate (A) and the presence of intradermal Zinc (B), here during a lichenoid
reaction on tattoo (images recorded at the Soleil Synchrotron, Diffabs line, by S. Reguer, D.
Bazin and H. Colboc).
The main objective of our project is to characterize the structural alterations of elastic
fibers during calcifying dermatosis.
The secondary objectives are:
- to study the consequences of cutaneous inflammatory processes on the alteration of
elastic fibers,
- to identify a possible nucleation foci for the formation of phospho-calcium deposits
within the elastic dermal and vascular fibers.
Skin samples. The study will be mainly performed on human skin, but also on a mouse model of
PXE: ABCC6 KO mouse. More precisely, the material studied at LOB will be:
- Human skin biopsies already collected for the diagnosis of these calcifying dermatoses:
PXE, calciphylaxis, other calcifying inflammatory dermatoses (lupus panniculitis,
dermatomyositis), from the pathology laboratory of the Tenon hospital (Dr Moguelet).
- As controls, human biopsies of healthy skin from patients of different ages, (margin of
skin tumour removal), from the Tenon Hospital. We will focuse on patient with advanced
skin aging, especially patients with solar elastosis. This model of UV induced elastic
fibers alteration is a great control in term of non-inflammatory and non-calcified
elastic fibers damages.
- Cutaneous biopsies from the mouse model of PXE (ABCC6 KO mouse) and from matched control
mice, from INSERM Unit 1155, Tenon Hospital.
Protocols. All these biopsies will be characterized by multiphoton microscopy by combining
2PEF and SHG contrasts in order to simultaneously image elastic and collagen fibers. In a
first step, the experimental protocol will be as follows:
- Production at Tenon Hospital of several serial slides from each biopsy: 1 unstained
("white") slide for multiphoton imaging, 1 slide with usual morphological staining
(hematoxylin-eosin-safran, HES), 1 slide with staining highlighting calcium deposits
(Von Kossa) and 3 slides with immuno-histochemical labelings of the main MMPs
(antibodies against MMP-1, 2 and 9).
- imaging at LOB on a standard brightfield optical microscope (standard and polarized
transmitted light, fluorescence, DIC) of all the stained slides over a large field of
view by mosaicking; identification of regions of interest (ROI) corresponding to
inflammatory processes, tissue remodeling or mineral deposits. Note that mineral
deposits are revealed by polarized transmitted light or by phase contrast or DIC.
- X-ray fluorescence imaging at Soleil Synchrotron to identify the metallic deposits
present in the biopsies, specially Zinc. This will be done in collaboration with
Dominique Bazin (Institute of Physico-Chemistry, CNRS, Paris-Saclay University), which
has a strong expertise in mineral imaging in the Diffabs line.
- Multiphoton imaging at LOB of the identified regions of interest, or even on a large
field of view by automatically stitching several tiles. Polarization-resolved images
will also be carried out in very dense areas or in remodeled areas in order to map the
direction of collagen and elastin fibers (10).
- Automated quantitative analysis of multiphoton LOB images to measure several parameters
related to the quantity and structure of elastin. Automated quantitative analyses will
also be performed on collagen and other structures of interest observed on multiphoton
images or histological images. The results of the different imaging modalities will be
correlated on each series of slides. Note that automatic analysis is essential both to
avoid the usual biases of semi-quantitative analysis and to obtain more informative
structural parameters. For instance, the following parameters will be extracted from the
elastin images: the mean density of the elastic fibers (per cm², in the papillary dermis
and in the reticular dermis), the orientation distribution of the elastic fibers, the
entropy and the circular variance of this distribution, the degree of curvature of the
elastic fibers, their distance from the mineral deposits.
- Statistical analysis of multimodal data, based on appropriate statistical tests. A
priori non-parametric tests (Wilcoxon-Mann-Whitney) will be used in view of the
relatively small number of available human samples. A minimum of 8 patients per group
will be studied, which should be sufficient due to the accuracy and reproducibility of
the automated measurements. If several samples or image series are performed on the same
patient, nested statistical tests will be used (nested t-tests or nested Wilcoxon).
In a second step and based on the results of the first step, various items of this protocol
will be improved to access more information:
- Multiphoton imaging will be performed in intact murine skin biopsies to better highlight
the 3D organization of elastin.
- Advanced fluorescence imaging of inflammation will be performed in ex vivo murine skin,
using spectral or FLIM (fluorescence lifetime imaging microscopy) measurements
- Third Harmonic Generation (THG) will be used to visualize the structure of the mineral
deposits.
1. Bazin D, et al. Characterization and some physicochemical aspects of pathological
microcalcifications. Chem Rev. 10 oct 2012;112(10):5092-120.
2. H Colboc, et al. Localization, morphologic features, and chemical composition of
calciphylaxis-related skin deposits in patients with calcific uremic
arteriolopathy. JAMA Dermatology 2019.
3. H Colboc, et al. Physicochemical characterization of inorganic deposits associated
with granulomas in cutaneous sarcoidosis. Journal of the European Academy of
Dermatology and Venereology 2018.
4. Haka AS, et al. Identifying microcalcifications in benign and malignant breast
lesions by probing differences in their chemical composition using Raman
spectroscopy. Cancer Res. 15 sept 2002;62(18):5375-80.
5. Munavalli G, et al. Weight loss-induced calciphylaxis: potential role of matrix
metalloproteinases. J Dermatol. 2003;30(12):915-919.
6. Schanne-Klein M-C. "L'imagerie multiphoton des peaux naturelles et synthétiques. Un
nouvel outil pour l'évaluation des produits cosmétiques." Photoniques 88 (2017):
21-24.
7. Wang, Hequn, et al. "Age-related morphological changes of the dermal matrix in
human skin documented in vivo by multiphoton microscopy." Journal of biomedical
optics 23.3 (2018): 030501.
8. Cui, Jason Z., et al. "Quantification of aortic and cutaneous elastin and collagen
morphology in Marfan syndrome by multiphoton microscopy." Journal of structural
biology 187.3 (2014): 242-253.
9. Tong, P. L., et al. "A quantitative approach to histopathological dissection of
elastin-related disorders using multiphoton microscopy." British Journal of
Dermatology 169.4 (2013): 869-879.
10. G. Ducourthial, J.-S. Affagard, M. Schmeltz, X. Solinas, M. Lopez-Poncelas, C.
Bonod-Bidaud, R. Rubio-Amador, F. Ruggiero, J.-M. Allain, E. Beaurepaire, and M.-C.
Schanne-Klein, "Monitoring dynamic collagen reorganization during skin stretching
with fast polarization-resolved SHG imaging.," J. Biophot. 12, e201800336 (2019).
