J Neuropathol Exp Neurol Copyright Ó 2014 by the American Association of Neuropathologists, Inc.
LETTER TO
Granular Osmiophilic MaterialYContaining Pseudoinclusions in CADASIL We recently described the role of electron microscopy examination in the diagnosis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome in a retrospective study of 13 cases (1). In 1 patient who was homozygous for the G528C mutation (2), vascular smooth muscle cells (VSMCs) showed several cytoplasmic inclusions that were found to be granular osmiophilic material (GOM)Y containing pseudoinclusions; the other 12 heterozygous patients did not seem to be affected. To determine whether GOMcontaining pseudoinclusions are a pathognomonic sign of CADASIL syndrome, particularly of the homozygous condition, we retrieved from our archives biopsy samples from all 13 patients (8 men and 5 women; age range, 45Y64 years; mean, 53 years) with a diagnosis of CADASIL disease and re-examined them by electron microscopy to assess GOM-containing pseudoinclusions both qualitatively and quantitatively. Patients are listed in the Table; age, sex, sample type, molecular findings, and genotype are presented by Morroni et al (1). Patient 12 was homozygous for the G528C mutation (2). Granular osmiophilic materialY containing pseudoinclusions were quantified by examining 50 nucleated VSMCs or VSMCs devoid of the nucleus but containing abundant cytoplasm in small and medium arteries of each patient sample and by calculating the proportion of those bearing GOM-containing pseudoinclusions. Vascular smooth muscle cell cytoplasmic processes were not included in the calculation. Each pseudoinclusion was surrounded by a cell membrane and separated from it by an electron-lucent halo (Fig. A). The cytoplasmic membrane showed numerous pinocytotic vesicles (Fig. A) that were sometimes arranged on the surface of the cell membrane, forming a sort of
Vol. 73, No. 9 September 2014 pp. 903Y905
THE
EDITOR
garland (Fig. B). In addition to GOM deposits, the larger pseudoinclusions also showed basal membrane and interstitium, sometimes containing collagen fibers (Fig. C). Some VSMCs contained more than 1 pseudoinclusion (from 1 to 3). The proportion of VSMCs containing pseudoinclusions correlated with the degree of cell branching, evaluated qualitatively by examining the surface of all arterioles in each sample (Table), and was highest (22% and 22.5%, respectively) in Cases 3 and 12, the latter being the homozygous patient. Both kidney and skin samples were examined in Patient 4. In renal arteries, GOM deposits were few and small; despite the extensive VSMC branching, they were not found in pseudoinclusions, whereas a large proportion (10%) of pseudoinclusions contained interstitium (basal membrane and collagen) but not GOM (Fig. D). In the skin sample, VSMCs exhibited more modest branching, and only 4% bore GOM-containing pseudoinclusions. The present findings document the common presence of GOM-containing pseudoinclusions in patients with CADASIL syndrome. They were detected in 9 of 13 cases (70%) in the tissue samples used for disease diagnosis; the renal sample was not included in the calculation (1, 3). The
proportion of VSMCs containing pseudoinclusions correlated with the extent of VSMC alteration, but not with patient genotype. Because they are extracellular, the GOM deposits found in pseudoinclusions were external to smooth muscle cells. In fact, a cytoplasmic pseudoinclusion does not lie free in the cytoplasm but is separated from it by invagination of the cytoplasmic membrane. An invagination in longitudinal section will be a pseudoinclusion in transverse section. Accordingly, GOM found in a deep membrane infolding appears as GOM-containing pseudoinclusions in transverse section (Fig. E). Large GOM-containing pseudoinclusions may encompass both the VSMC basal membrane and collagen fibers. The abovementioned considerations clarify the correlation between GOMcontaining pseudoinclusions and VSMC branching (i.e. the more irregular and infolded is the VSMC membrane, the larger is the proportion of VSMCs showing GOM-containing pseudoinclusions). Excluding the renal sample (Patient 4), the proportion of VSMCs bearing GOMcontaining pseudoinclusions was largest in the 6 patients with extensive VSMC branching.
TABLE. Extent of VSMC Branching and Proportion of Cells Showing GOM-Containing Pseudoinclusions in 13 CADASIL Patients Patient 1 2 3 4 5 6 7 8 9 10 11 12 13
Branching
VSMCs Bearing GOM-Containing Pseudoinclusions, %
+ +++ +++ + (Skin) +++ (Kidney) +++ +++ + + + + +++ +++ +
0 17.5 22 4 0 12 8.5 0 0 0 2 10 22.5 4
(+) Light, (++) moderate; (+++) extensive.
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
903
Letter to the Editor
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
FIGURE. (A) Transmission electron microscopy of a skin biopsy (Case 12). Two GOM-containing pseudoinclusions (pse) within a VSMC are shown. The cell surface has an irregular profile (arrowheads). Inset: Enlargement of the boxed area. (B) Transmission electron microscopy of a skin biopsy (Case 13). Three GOM deposits are depicted: 2 on the surface of the VSMC and 1 contained in a pse. The cell nucleus is very irregular. Inset: Enlargement of the boxed area showing numerous pinocytotic vesicles arranged as a garland close to the cytoplasmic membrane of the pse. En, endothelium. (C) Transmission electron microscopy of a skin biopsy (Case 2). Granular osmiophilic material deposits on the surface of a VSMC and a GOM-containing pse. The basal membrane (bm) within the pse is also recognizable. (D) Transmission electron microscopy of a kidney biopsy (Case 4). This VSMC shows a GOM, 2 pse containing bm and collagen but not GOM, and a deep invagination (in). A GOM deposit is also evident at the top of the in. (E, a) Drawing of a smooth muscle cell exhibiting a deep indentation containing GOM. (E, b) In transverse section (dotted line), the indentation containing GOM gives rise to a pse. The vesicles close to the cell membrane are pinocytotic vesicles. bm, basal membrane; db, dense bodies; mt, mitochondrion. Scale bars = (A) 600 nm; inset, 300 nm; (B) 400 nm; inset, 200 nm; (C) 700 nm; (D) 750 nm.
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Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
To our knowledge, GOM-containing pseudoinclusions were first described by our group in a skin biopsy sample from a homozygous CADASIL patient (Patient 12) (1, 2). This is the only patient with this genotype, but the phenotype of this patient did not differ from that of heterozygous patients (2). Here we demonstrate that GOM-containing pseudoinclusions are also found in VSMCs from heterozygous CADASIL patients. The fact that we missed these structures in heterozygous patients in the previous study can be explained as follows: i) we described GOM-containing pseudoinclusions in the 12th CADASIL case that came to our attention (Table) (1); because the cases are listed in chronologic order, Patient 12 was the next-to-the-last patient to be examined, which can be viewed as a sort of learning curve; ii) our interest in the disease was initially merely diagnosticV scientific interest was aroused later; and, finally, iii) some samples contained very few pseudoinclusions. In Patient 12, greater experience, increased scientific interest, and a high rate of VSMCs (22.5%) bearing GOM-containing pseudoinclusions led to their identification and to the first report. Ishiko et al (4) described electrondense material continuous with the plasma membrane in the VSMC cytoplasm. Even though the image that they provided is very small, the structure depicted is consistent with a GOM-containing pseudoinclusion. In CADASIL transgenic mice, Arboleda-Velasquez et al (5) described osmiophilic deposits associated with vesicles and inclusions in VSMCs of brain vessels but provided no images. Yamamoto et al (6) also showed a perivascular cell (which they interpreted as a phagocyteappearing cell) in human meningeal vessels that seemed to engulf GOM deposits; we
interpreted it as a VSMC sectioned tangentially (7) and considered the findings reported by Arboleda-Velasquez et al (5) and Yamamoto et al (6) as pseudoinclusions as well. Our current data demonstrate that GOM-containing pseudoinclusions could be ultrastructural markers for CADASIL, that they are independent of genotype, and that they correlate with VSMC morphologic changes. Manrico Morroni, MD, BSc Section of Neuroscience and Cell Biology Department of Experimental and Clinical Medicine School of Medicine Universita` Politecnica delle Marche Ancona, Italy Electron Microscopy Unit United Hospitals Ancona, Italy
Teresa Lorenzi, PhD and Mario Castellucci, PhD, MD Section of Neuroscience and Cell Biology Department of Experimental and Clinical Medicine School of Medicine Universita` Politecnica delle Marche Ancona, Italy
Michele Ragno, MD Division of Neurology Mazzoni Hospital Ascoli Piceno, Italy
Marina Scarpelli, MD Section of Pathological Anatomy Department of Biomedical Sciences and Public Health School of Medicine Universita` Politecnica delle Marche United Hospitals Ancona, Italy
Letter to the Editor
Sources of support: The study was supported by a grant from Universita` Politecnica delle Marche (2013 FAR, formerly 60%) to Manrico Morroni. ACKNOWLEDGMENTS We are grateful to Maria Cristina Zingaretti and Michela Cardinali for their technical assistance and to Word Designs (www.silviamodena.com) for language revision.
REFERENCES 1. Morroni M, Marzioni D, Ragno M, et al. Role of electron microscopy in the diagnosis of CADASIL syndrome: A study of 32 patients. PLoS One 2013; 8:e65482 2. Ragno M, Pianese L, Morroni M, et al. ‘‘CADASIL coma’’ in an Italian homozygous CADASIL patient: Comparison with clinical and MRI findings in age-matched heterozygous patients with the same G528C NOTCH3 mutation. Neurol Sci 2013;34: 1947Y53 3. Ragno M, Trojano L, Pianese L, et al. Renal involvement in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL): Report of a case with a six-year follow-up. Histol Histopathol 2012;27:1307Y14 4. Ishiko A, Shimizu A, Nagata E, et al. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Am J Dermatopathol 2005;27:131Y34 5. Arboleda-Velasquez JF, Manent J, Lee JH, et al. Hypomorphic Notch3 alleles link Notch signaling to ischemic cerebral small-vessel disease. Proc Natl Acad Sci U S A 2011;108:E128Y35 6. Yamamoto Y, Craggs LJ, Watanabe A, et al. Brain microvascular accumulation and distribution of the NOTCH3 ectodomain and granular osmiophilic material in CADASIL. J Neuropathol Exp Neurol 2013;72:416Y31 7. Morroni M, Lorenzi T. CADASIL: Intracytoplasmic GOM deposits are pseudoinclusions. J Neuropathol Exp Neurol 2013;72:801
Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
905
LETTER TO
Granular Osmiophilic MaterialYContaining Pseudoinclusions in CADASIL We recently described the role of electron microscopy examination in the diagnosis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome in a retrospective study of 13 cases (1). In 1 patient who was homozygous for the G528C mutation (2), vascular smooth muscle cells (VSMCs) showed several cytoplasmic inclusions that were found to be granular osmiophilic material (GOM)Y containing pseudoinclusions; the other 12 heterozygous patients did not seem to be affected. To determine whether GOMcontaining pseudoinclusions are a pathognomonic sign of CADASIL syndrome, particularly of the homozygous condition, we retrieved from our archives biopsy samples from all 13 patients (8 men and 5 women; age range, 45Y64 years; mean, 53 years) with a diagnosis of CADASIL disease and re-examined them by electron microscopy to assess GOM-containing pseudoinclusions both qualitatively and quantitatively. Patients are listed in the Table; age, sex, sample type, molecular findings, and genotype are presented by Morroni et al (1). Patient 12 was homozygous for the G528C mutation (2). Granular osmiophilic materialY containing pseudoinclusions were quantified by examining 50 nucleated VSMCs or VSMCs devoid of the nucleus but containing abundant cytoplasm in small and medium arteries of each patient sample and by calculating the proportion of those bearing GOM-containing pseudoinclusions. Vascular smooth muscle cell cytoplasmic processes were not included in the calculation. Each pseudoinclusion was surrounded by a cell membrane and separated from it by an electron-lucent halo (Fig. A). The cytoplasmic membrane showed numerous pinocytotic vesicles (Fig. A) that were sometimes arranged on the surface of the cell membrane, forming a sort of
Vol. 73, No. 9 September 2014 pp. 903Y905
THE
EDITOR
garland (Fig. B). In addition to GOM deposits, the larger pseudoinclusions also showed basal membrane and interstitium, sometimes containing collagen fibers (Fig. C). Some VSMCs contained more than 1 pseudoinclusion (from 1 to 3). The proportion of VSMCs containing pseudoinclusions correlated with the degree of cell branching, evaluated qualitatively by examining the surface of all arterioles in each sample (Table), and was highest (22% and 22.5%, respectively) in Cases 3 and 12, the latter being the homozygous patient. Both kidney and skin samples were examined in Patient 4. In renal arteries, GOM deposits were few and small; despite the extensive VSMC branching, they were not found in pseudoinclusions, whereas a large proportion (10%) of pseudoinclusions contained interstitium (basal membrane and collagen) but not GOM (Fig. D). In the skin sample, VSMCs exhibited more modest branching, and only 4% bore GOM-containing pseudoinclusions. The present findings document the common presence of GOM-containing pseudoinclusions in patients with CADASIL syndrome. They were detected in 9 of 13 cases (70%) in the tissue samples used for disease diagnosis; the renal sample was not included in the calculation (1, 3). The
proportion of VSMCs containing pseudoinclusions correlated with the extent of VSMC alteration, but not with patient genotype. Because they are extracellular, the GOM deposits found in pseudoinclusions were external to smooth muscle cells. In fact, a cytoplasmic pseudoinclusion does not lie free in the cytoplasm but is separated from it by invagination of the cytoplasmic membrane. An invagination in longitudinal section will be a pseudoinclusion in transverse section. Accordingly, GOM found in a deep membrane infolding appears as GOM-containing pseudoinclusions in transverse section (Fig. E). Large GOM-containing pseudoinclusions may encompass both the VSMC basal membrane and collagen fibers. The abovementioned considerations clarify the correlation between GOMcontaining pseudoinclusions and VSMC branching (i.e. the more irregular and infolded is the VSMC membrane, the larger is the proportion of VSMCs showing GOM-containing pseudoinclusions). Excluding the renal sample (Patient 4), the proportion of VSMCs bearing GOMcontaining pseudoinclusions was largest in the 6 patients with extensive VSMC branching.
TABLE. Extent of VSMC Branching and Proportion of Cells Showing GOM-Containing Pseudoinclusions in 13 CADASIL Patients Patient 1 2 3 4 5 6 7 8 9 10 11 12 13
Branching
VSMCs Bearing GOM-Containing Pseudoinclusions, %
+ +++ +++ + (Skin) +++ (Kidney) +++ +++ + + + + +++ +++ +
0 17.5 22 4 0 12 8.5 0 0 0 2 10 22.5 4
(+) Light, (++) moderate; (+++) extensive.
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
903
Letter to the Editor
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
FIGURE. (A) Transmission electron microscopy of a skin biopsy (Case 12). Two GOM-containing pseudoinclusions (pse) within a VSMC are shown. The cell surface has an irregular profile (arrowheads). Inset: Enlargement of the boxed area. (B) Transmission electron microscopy of a skin biopsy (Case 13). Three GOM deposits are depicted: 2 on the surface of the VSMC and 1 contained in a pse. The cell nucleus is very irregular. Inset: Enlargement of the boxed area showing numerous pinocytotic vesicles arranged as a garland close to the cytoplasmic membrane of the pse. En, endothelium. (C) Transmission electron microscopy of a skin biopsy (Case 2). Granular osmiophilic material deposits on the surface of a VSMC and a GOM-containing pse. The basal membrane (bm) within the pse is also recognizable. (D) Transmission electron microscopy of a kidney biopsy (Case 4). This VSMC shows a GOM, 2 pse containing bm and collagen but not GOM, and a deep invagination (in). A GOM deposit is also evident at the top of the in. (E, a) Drawing of a smooth muscle cell exhibiting a deep indentation containing GOM. (E, b) In transverse section (dotted line), the indentation containing GOM gives rise to a pse. The vesicles close to the cell membrane are pinocytotic vesicles. bm, basal membrane; db, dense bodies; mt, mitochondrion. Scale bars = (A) 600 nm; inset, 300 nm; (B) 400 nm; inset, 200 nm; (C) 700 nm; (D) 750 nm.
904
Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 73, Number 9, September 2014
To our knowledge, GOM-containing pseudoinclusions were first described by our group in a skin biopsy sample from a homozygous CADASIL patient (Patient 12) (1, 2). This is the only patient with this genotype, but the phenotype of this patient did not differ from that of heterozygous patients (2). Here we demonstrate that GOM-containing pseudoinclusions are also found in VSMCs from heterozygous CADASIL patients. The fact that we missed these structures in heterozygous patients in the previous study can be explained as follows: i) we described GOM-containing pseudoinclusions in the 12th CADASIL case that came to our attention (Table) (1); because the cases are listed in chronologic order, Patient 12 was the next-to-the-last patient to be examined, which can be viewed as a sort of learning curve; ii) our interest in the disease was initially merely diagnosticV scientific interest was aroused later; and, finally, iii) some samples contained very few pseudoinclusions. In Patient 12, greater experience, increased scientific interest, and a high rate of VSMCs (22.5%) bearing GOM-containing pseudoinclusions led to their identification and to the first report. Ishiko et al (4) described electrondense material continuous with the plasma membrane in the VSMC cytoplasm. Even though the image that they provided is very small, the structure depicted is consistent with a GOM-containing pseudoinclusion. In CADASIL transgenic mice, Arboleda-Velasquez et al (5) described osmiophilic deposits associated with vesicles and inclusions in VSMCs of brain vessels but provided no images. Yamamoto et al (6) also showed a perivascular cell (which they interpreted as a phagocyteappearing cell) in human meningeal vessels that seemed to engulf GOM deposits; we
interpreted it as a VSMC sectioned tangentially (7) and considered the findings reported by Arboleda-Velasquez et al (5) and Yamamoto et al (6) as pseudoinclusions as well. Our current data demonstrate that GOM-containing pseudoinclusions could be ultrastructural markers for CADASIL, that they are independent of genotype, and that they correlate with VSMC morphologic changes. Manrico Morroni, MD, BSc Section of Neuroscience and Cell Biology Department of Experimental and Clinical Medicine School of Medicine Universita` Politecnica delle Marche Ancona, Italy Electron Microscopy Unit United Hospitals Ancona, Italy
Teresa Lorenzi, PhD and Mario Castellucci, PhD, MD Section of Neuroscience and Cell Biology Department of Experimental and Clinical Medicine School of Medicine Universita` Politecnica delle Marche Ancona, Italy
Michele Ragno, MD Division of Neurology Mazzoni Hospital Ascoli Piceno, Italy
Marina Scarpelli, MD Section of Pathological Anatomy Department of Biomedical Sciences and Public Health School of Medicine Universita` Politecnica delle Marche United Hospitals Ancona, Italy
Letter to the Editor
Sources of support: The study was supported by a grant from Universita` Politecnica delle Marche (2013 FAR, formerly 60%) to Manrico Morroni. ACKNOWLEDGMENTS We are grateful to Maria Cristina Zingaretti and Michela Cardinali for their technical assistance and to Word Designs (www.silviamodena.com) for language revision.
REFERENCES 1. Morroni M, Marzioni D, Ragno M, et al. Role of electron microscopy in the diagnosis of CADASIL syndrome: A study of 32 patients. PLoS One 2013; 8:e65482 2. Ragno M, Pianese L, Morroni M, et al. ‘‘CADASIL coma’’ in an Italian homozygous CADASIL patient: Comparison with clinical and MRI findings in age-matched heterozygous patients with the same G528C NOTCH3 mutation. Neurol Sci 2013;34: 1947Y53 3. Ragno M, Trojano L, Pianese L, et al. Renal involvement in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL): Report of a case with a six-year follow-up. Histol Histopathol 2012;27:1307Y14 4. Ishiko A, Shimizu A, Nagata E, et al. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Am J Dermatopathol 2005;27:131Y34 5. Arboleda-Velasquez JF, Manent J, Lee JH, et al. Hypomorphic Notch3 alleles link Notch signaling to ischemic cerebral small-vessel disease. Proc Natl Acad Sci U S A 2011;108:E128Y35 6. Yamamoto Y, Craggs LJ, Watanabe A, et al. Brain microvascular accumulation and distribution of the NOTCH3 ectodomain and granular osmiophilic material in CADASIL. J Neuropathol Exp Neurol 2013;72:416Y31 7. Morroni M, Lorenzi T. CADASIL: Intracytoplasmic GOM deposits are pseudoinclusions. J Neuropathol Exp Neurol 2013;72:801
Ó 2014 American Association of Neuropathologists, Inc.
Copyright © 2014 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
905
