The exosomes isolated from serum of patients with systemic sclerosis contained higher levels of profibrotic miRNAs and lower levels of antifibrotic miRNAs compared to exosomes from healthy individuals. When applied to normal dermal fibroblasts in culture, the scleroderma patient exosomes stimulated the expression of profibrotic genes, mimicking the phenotype of scleroderma fibroblasts. This effect was partially reduced when the exosomes were pre-treated to degrade either the RNA or protein content, indicating both components contribute to the exosomes' ability to induce a profibrotic phenotype in target cells and suggesting a potential mechanism for the transmission and progression of fibrosis in scleroderma.

1. RNA AND PROTEIN CARGO OF EXOSOMES ISOLATED FROM SERUM OF SYSTEMIC SCLEROSIS PATIENTS
INDUCE A PROFIBROTIC PHENOTYPE IN CULTURED NORMAL HUMAN DERMAL FIBROBLASTS: A POTENTIAL
MECHANISM FOR THE INITIATION AND PROGRESSION OF A PROFIBROTIC PHENOTYPE IN SSc
CONCLUSIONS: The content of profibrotic/antifibrotic miRNA of serum exosomes from SSc patients is different from that of normal serum exosomes and displays an overall profibrotic phenotype. SSc patient-
derived exosomes induce a profibrotic phenotype in target cultured normal dermal fibroblasts. The induction of this profibrotic phenotype is mediated by both the RNA and protein content of the exosomes. Since
exosomes are involved in intercellular communication these observations suggest a mechanism for transmission of a profibrotic molecular program to normal target cells leading to the extension of the fibrotic
process to non-affected tissues. (Supported by NIH Grant AR055660 to SAJ.)
Peter. J. Wermuth, Ph.D., Kellan R. Carney, B.S., Sonsoles Piera-Velazquez, Ph.D., and Sergio. A. Jimenez, M.D.
Jefferson Institute of Molecular Medicine and Division of Connective Tissue Diseases and Scleroderma Center, Thomas Jefferson University, Philadelphia, Pa.,USA.
ABSTRACT
Background/Purpose: Exosomes are lipid bilayer-bound
microvesicles that contain various macromolecules including numerous
microRNA (miRNA) and proteins. Exosomes mediate intercellular
communication by fusing and releasing their macromolecular content
into target cells. The mechanism of the establishment and progression of
a profibrotic phenotype in Systemic Sclerosis (SSc) is not currently well
understood. Here, we characterized the miRNA content of exosomes
isolated from the serum of SSc patients and analyzed the ability of
exosomal RNA and protein components to induce a profibrotic
phenotype in normal human dermal fibroblasts in vitro.
Methods: Exosomes were isolated from serum from normal individuals
and from patients with either limited cutaneous or diffuse cutaneous SSc
employing a highly specific polymer precipitation procedure. The isolated
exosomes were characterized by Nanosight Particle Tracking Analysis and
transmission electron microscopy and the levels of nine pro-fibrotic and
nineteen antifibrotic miRNA were assessed by semiquantitative real time
PCR. Cultured normal human dermal fibroblasts were incubated with
untreated exosomes isolated from the serum of SSc patients or normal
donors or with isolated exosomes previously treated with Triton X-100, or
with RNAseA, or proteinase K, alone or in combination with Triton X-100
to selectively deplete miRNAs or proteins, respectively. The expression
levels of several profibrotic genes in the dermal fibroblasts treated with
exosomes isolated from serum of normal individuals or SSc patients were
assessed.
Results: Nanosight particle tracking analysis and transmission electron
microscopy confirmed the isolation of microvesicles in the size range
expected for exosomes and an exosome protein array verified that the
isolated particles contained exosome-specific proteins and were not
contaminated by Golgi-associated proteins. The content of six antifibrotic
miRNAs was decreased whereas the content of three profibrotic miRNAs
was increased in serum exosomes from both groups of SSc patients
compared to normal serum exosomes. Furthermore, the levels of four
miRNA were significantly different between the two SSc clinical subsets.
Untreated exosomes isolated from the serum of patients with limited or
diffuse SSc induced the expression of profibrotic genes in cultured normal
human dermal fibroblasts whereas the establishment of this profibrotic
phenotype could be partially abrogated by treatment of the exosomes
with either RNAse A or proteinase K.
Conclusions: The content of profibrotic/antifibrotic miRNA of serum
exosomes from SSc patients is different from that of normal serum
exosomes and displays an overall profibrotic phenotype. SSc patient-
derived exosomes can induce a profibrotic phenotype in target cultured
normal dermal fibroblasts. The induction of this profibrotic phenotype is
mediated by both the RNA and protein content of the exosomes. Since
exosomes are involved in intercellular communication these observations
suggest a mechanism for transmission of a profibrotic molecular program
to normal target cells leading to the extension of the fibrotic process to
non-affected tissues.
(Supported by NIH Grant AR055660 to SAJ.)
(A) Profibrotic miRNAs with a 2-fold or greater difference in content level in one or both
disease subgroups. (B) Antifibrotic miRNAs with a 2-fold or greater difference in content level
in one or both disease subgroups. (C) Antifibrotic miRNAs displaying content levels that were
significantly different between lcSSc and dcSSc subgroups.
Triplicate wells of normal fibroblasts were treated with one of three concentrations of
exosomes for each treatment group (N = 2 samples; L and D = 3 samples). Values
represent the average change in the levels of gene expression of (A) COL1A1, COL3A1
and FN1; (B) CTGF and TGF-β; and (C) FN-EDA, α-SMA and COMP. Expression levels
from the saline-treated fibroblasts were arbitrarily set at the 100% expression level.
Values for other samples are expressed relative to this control. Significance was
determined by Student’s T-test. Statistical significance: **: p<0.01;
***: p<0.001.
Equivalent amounts of exosomes were pre-treated with
either saline alone, 0.1% Triton-X100 alone, 20 μg/mL RNase
A alone, 100 μg/mL Proteinase K alone, or with 0.1% Triton-
X100 + 20 μg/mL Rnase A, or with 0.1% Triton-X100 + 100
μg/mL Proteinase K for 1 hr at 37o C followed by heat
inactivation of the enzymes and precipitation with ExoQuick
before being added to cultured normal human dermal
fibroblasts for 72 h.
B
Average change in the levels of gene expression of COL1A1 (A), COL3A1 (B), FN1 (C) and FN-EDA (D). Triplicate wells of normal
fibroblasts were treated with saline, or with 10 mg exosomes (based on protein concentration) of normal (N = 2 samples) or diffuse SSc
(D = 2 samples) exosomes pre-treated with either saline alone (U), Triton-X100 alone (T), RNase A alone (R), Proteinase K alone (P), or
with Triton-X100 + Rnase A (T+R) or with Triton-X100 + Proteinase K (T+P). Expression levels from the saline-treated fibroblasts (U)
were arbitrarily set at the 100% expression level. Values for N and D samples were averaged and are expressed relative to this control.
Significance was determined by Student’s T-test. Statistical significance: **: p<0.01; ***: p<0.001.
A
B
C
Results:
 The isolated exosomes were of the
expected size and shape.
 The content of six profibrotic miRNAs
was greater than 2-fold higher whereas
the content of ten antifibrotic miRNAs
was at least 2-fold lower in SSc
exosomes.
 The levels of eight miRNA were
significantly different between the two
SSc clinical subsets compared to
exosomes from normal serum.
 Exosomes isolated from SSc patient
serum caused a dose related stimulation
of profibrotic gene expression in normal
dermal fibroblasts, raising them to the
levels normally seen in SSc fibroblasts.
(A) Size distribution and concentration of purified exosomes. (B) Transmission electron microscopy.
Larger vesicles (large arrows) of ~100-120 nm size are interspersed with a population of smaller vesicles
(small arrows) of ~40-60 nm (left panel). Enlarged electron micrograph of a representative 100 nm
exosome. Scale bars = 100 nm (right panel). (C) Exosome antibody membrane arrays confirming the
presence of the exosome-specific proteins CD63, CD81, ALIX, ANXA5 and TSG101 and the absence of
the cytosolic cis-Golgi protein GM130.
Introduction
 Exosomes are lipid bilayer-bound
microvesicles that contain various
macromolecules including numerous
microRNA (miRNA) and proteins.
 Exosomes mediate intercellular
communication by fusing and releasing
their macromolecular content into
target cells.
 The mechanism of the establishment
and progression of a profibrotic
phenotype in Systemic Sclerosis (SSc) is
not currently well understood.
 The miRNA contained in blood serum
of normal individuals and SSc patients
was characterized and their ability to
induce a profibrotic phenotype in
target fibroblasts was analyzed.
Content levels of miRNA present in exosomes isolated from serum
of donors with limited and diffuse SSc as assessed by real time PCR.
Measurements of particle size and concentration of exosomes isolated
from the serum of patients with limited and diffuse cutaneous SSc.
Effects of SSc exosomes on the expression of
extracellular matrix components, growth factors and
myofibroblast-specific genes in cultured normal human
dermal fibroblasts.
A B
C D
Effects of pre-treatment of SSc exosomes on their ability to modify the phenotype of
target cultured normal human dermal fibroblasts.
Strategy for pre-treatment of exosomes for
the analysis of effect of exosome RNA and
protein components on exosome-mediated
alteration of target cell phenotype.