Johnny L. Efendy

Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body

This study utilized both in vivo and in vitro techniques to investigate whether cells of bone marrow origin can differentiate into smooth muscle-like cells (myofibroblasts) with contractile filaments and proteins. Female C57BL/6 mice expressing the Ly5.2 antigen on the surface of their haemopoietic cells had four pieces of silastic tubing (3 × 0.5 mm outer diameter) or boiled blood clot (2–3 mm diameter) placed in their peritoneal cavity. After 3, 5, 7 and 14 days (n = 4/group) the implants were removed and those that had remained free-floating were processed for light microscopy, immunohistochemistry and electron microscopy. In the first 3–5 days, rounded cells adhered to the entire surface of the tubing then flattened. These cells stained with fluoresceinated antibodies to Ly5.2 showing that they were derived from haemopoietic cells. By 14 days the cells had become elongated and multilayered in a collagen matrix, forming a thick tissue capsule around the tubing or boiled clot. They contained contractile filaments and stained with antibodies to α-smooth muscle actin but no longer stained for Ly5.2. A separate set of female C57BL/6 Ly5.2 mice were X-irradiated to destroy bone marrow then immediately transfused with 106 nucleated bone marrow cells taken from the femur and tibia of a congenic strain of male mice expressing the Ly5.1 allele. Eight of the female mice with successful engraftment (80–99%) had silastic tubing implanted in the peritoneal cavity. After 14 days, in situ hybridization with Y chromosome probe confirmed the male donor, and thus bone marrow, origin of the elongated cells that formed the capsule. In vitro studies showed that cells of the murine macrophage cell lines RAW 264.7 and J774 express α-smooth muscle actin after exposure to the cytokine γ-interferon in vitro. These data show that bone marrow-derived cells can differentiate into smooth muscle-like cells and raises the possibility that blood-derived cells may contribute to the development of fibro-proliferative vascular diseases such as atherosclerosis.

Blood vessels from bone marrow

Abstract: Lengths of silastic tubing were inserted into the peritoneal cavity of rats or rabbits. By two weeks the free‐floating implants had become covered by a capsule consisting of several layers of “macrophage”‐derived myofibroblasts and collagen matrix overlaid by a single layer of mesothelial cells. The tubing was removed from the harvested implant and the tissue everted. This now resembled an artery with an inner lining of mesothelial cells (the “intima”), a “media” of myofibroblasts, and an outer collagenous “adventitia.” The tube of living tissue was grafted by end‐to‐end anastomoses into the transected carotid artery or abdominal aorta of the same animal in which the tissue had been grown, where it remained patent for four months and developed structures resembling elastic lamellae. The myofibroblasts developed a high volume fraction of myofilaments and became responsive to contractile and relaxing agents similar to smooth muscle cells of the adjacent artery wall.

The effect of environmental cues on the differentiation of myofibroblasts in peritoneal granulation tissue

This study investigated the effect of haemodynamic stress, active stretch, and neuronal input on the differentiation of myofibroblasts in peritoneal granulation tissue. Lengths of silastic tubing (10 mm long×3 mm diameter) were placed in the peritoneal cavity of the rat. By 2 weeks, a capsule of granulation tissue had formed around the tubing. This capsule consisted of several layers of myofibroblasts and the matrix that they had produced, overlaid by a single layer of mesothelial cells. The silastic tubing was removed and at the same time, the living tube of tissue was everted so that the mesothelium now lined its inner surface. To examine the effect of haemodynamic factors on myofibroblast differentiation, the 10 mm long tubes of mesothelial‐lined granulation tissue were transplanted into the severed abdominal aorta of the same rat in which the granulation tissue was grown. End‐to‐end anastomoses were performed to extend the existing aorta. At 1, 2, and 3 months post‐transplantation, the grafts were removed and a progressive increase in the percent volume fraction of myofilaments (% Vvmyo) was observed (from 35.7±1.6% to 58.7 3±1.4%; p<0.05). To determine whether the active stretching that occurs in vivo could account for differentiation of the constituent myofibroblasts, tubes of granulation tissue were placed into a mechanical device in which they underwent continuous stretching of 5–10% elongation from the resting position at 50 cycles per minute for 3, 24 or 72 h. This caused a significant (p<0.05) increase in %Vvmyo after 72 h. Granulation tissue was also transplanted into the rat anterior eye chamber, where it became surrounded by adrenergic nerves supplying the host iris. Two months after implantation, there was no significant change in the %Vvmyo of the myofibroblasts (35.7±1.6% to 33.3±2.7%). These studies show that myofibroblasts of the granulation tissue encapsulating free‐floating foreign bodies in the peritoneal cavity further differentiate towards a smooth muscle phenotype when transplanted into a smooth muscle environment, namely the abdominal aorta. Similar changes are seen when the granulation tissue is subjected to active, intermittent stretch in vitro, while the presence of nerves has no effect. Copyright

Vascular Smooth Muscle Cells

Smooth muscle is present in one form or another in most organs, and its contractile activity is vital for normal functioning of the body. To be able to perform the multitude of tasks required of the organs, smooth muscle cells vary widely in their patterns of activity. In blood vessels, a continuous and maintained activity is required, whereas in the uterus only occasional bursts of activity are needed. Some tissues act as a unit (urinary bladder), whereas others have localized contraction (arterioles) or a peristaltic wave of contraction passing through them (intestine) [1, 2]. Functionally, smooth muscle can be broadly classified into tonic and phasic types [2–4]. Tonic smooth muscle cells do not normally generate action potentials. They have a relatively high content of the LC-17b isoform of the alkali myosin light chain, and have myosin heavy chains that lack a 7-amino-acid insert. These slow myosin isoforms have a higher affinity for Mg++ ADP and this may contribute to the “latch” state and slow shortening velocity [4, 5].