Ira Mills

Molecular basis for tissue expansion : Clinical implications for the surgeon

A wide variety of tissue expansion techniques have been used for breast reconstruction, craniofacial surgery, and burn care in plastic reconstructive surgery. However, the basic mechanism by which skin and surrounding tissue respond to mechanical expansion remains unclear. Recent studies have revealed the biomechanical aspects of cells subjected to strain and various factors involved in the stretch-induced signal transduction pathway. In this regard, we have reported previously that mechanical force increases keratinocyte growth and protein synthesis and alters cell morphology. The mechanism by which strain causes an enhancement of cellular growth appears to be a network of several integrated cascades, implicating growth factors, cytoskeleton, and the protein kinase family. Recently, additional evidence has accumulated that mechanical strain stimulates signal transduction pathways that could trigger a series of cascades eventually leading to a new skin production. For example, we have evidence suggesting a key role for protein kinase C (PKC) in mechanosignaling as PKC is activated and translocated in keratinocytes subjected to strain in an isoform-specific manner. In this report, molecular mechanisms leading to enhancement of skin surface area are reviewed, and possible future applications are discussed.

Gene regulation by mechanical forces

Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

Cultured vascular smooth muscle cells from porcine coronary artery possess A1 and A2 adenosine receptor activity

This study tested the hypothesis that an A1 adenosine receptor capable of inhibiting adenylate cyclase activity is present in porcine coronary vascular smooth muscle cells. In the absence of blockade of the A2 adenosine receptor, the A1 adenosine receptor agonists phenylisopropyladenosine (PIA) and cyclopentyladenosine (CPA) (10(-9) M) failed to inhibit Gpp(NH)p stimulated adenylate cyclase activity. However, after blockade of the A2 adenosine receptor with 30 nM CGS 15943A, cyclopentyladenosine (10(-9) M) inhibited Gpp(NH)p stimulated adenylate cyclase activity by 27 +/- 3% (4.3 +/- 0.7, Mean +/- SEM; pmoles/min/mg vs 5.9 +/- 0.8, P less than .05). The data demonstrate that both A1 and A2 adenosine receptors are present in coronary vascular smooth muscle. The results indicate that adenosine may mediate both vasodilation and vasoconstriction in the coronary circulation via A2 and A1 adenosine receptors, respectively.

Cyclic Strain Stimulates Endothelial Cell Proliferation: Characterization of Strain Requirements

Previous studies from our laboratory show that vascular cell biology can be modulated by cyclic strain. However, the precise relationship between cyclic strain and endothelial cell responsiveness remains unclear. The objective of this study was to better characterize this relationship by examining the proliferative response of endothelial cells to sharply defined ranges of strain. This was accomplished by seeding bovine aortic endothelial cells (passages 1–3; N = 9) (10,000 cells/cm2) either on the outer 2/3 (periphery) or the inner 1/3 (center) regions of collagen-coated flexible bottomed wells. The wells were deformed at a frequency of 100 cycles per minute with either -5 kPa vacuum (center, 0–3.2% strain; periphery, 3.2–11% strain) or -20 kPa vacuum (center, 0.37–6.3% strain; periphery, 6.3–25% strain) for up to 5 days. Static, unstretched wells served as controls. At 5 days, endothelial cells subjected to either low (-5 kPa) or high vacuum (-20 kPa), exhibited a stimulated proliferative response in bo...

Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.

Metabolic effects of β, α1, and α2 adrenoceptor activation on Brown adipocytes isolated from the perirenal adipose tissue of fetal lambs

Abstract Brown adipocytes can be readily isolated by collagenase digestion of perirenal adipose tissue from fetal lambs. In isolated cells the addition of phenylephrine in the presence of alprenolol (to specifically stimulate α adrenoceptors) resulted in an increase in de novo synthesis of phosphatidylinositol and phosphatidic acid. The stimulatory effects were preferentially inhibited by prazosin while yohimbine had little effect, indicating that the adrenoceptors were α 1 in character. Isoproterenol stimulated cyclic adenosine monophosphate (AMP) accumulation and lipolysis as well as respiration. Forskolin also mimicked the effects of β adrenergic stimulation. Clonidine, a specific α 2 adrenergic agonist, inhibited lipolysis and cyclic AMP accumulation. Insulin inhibited cyclic AMP accumulation and stimulated glucose metabolism in the adipocytes. The present studies indicate that β, α 1 , and α 2 adrenergic as well as insulin responses can be detected in ovine perirenal adipocytes.

Pertussis toxin reversal of the antilipolytic action of insulin in rat adipocytes in the presence of forskolin does not involve cyclic AMP

Insulin inhibition of lipolysis in the presence of forskolin was reversed by a four hour exposure of adipocytes to pertussis toxin. In contrast, the antilipolytic action of insulin against lipolysis due to theophylline was unaffected by pertussis toxin as was the ability of insulin to lower cyclic AMP in the presence of either forskolin or theophylline. The stimulation of adenylate cyclase by norepinephrine in crude plasma membranes obtained from rat adipocytes was inhibited by N6-(Phenylisopropyl)adenosine (PIA) and abolished by pretreating rat adipocytes with pertussis toxin. The stimulation of glucose metabolism by insulin was not altered by pertussis toxin pretreatment of rat adipocytes. These findings suggest that pertussis toxin selectively abolishes the antilipolytic effect of insulin in the presence of forskolin through a cyclic AMP independent mechanism.

Activation of lipolysis and cyclic AMP accumulation in rabbit adipocytes by isoproterenol in the presence of forskolin or pertussis toxin

Adipocytes from rabbits are relatively insensitive to catecholamines or forskolin. However, the combination of catecholamines plus forskolin increased cyclic AMP accumulation and lipolysis much more than either agent alone. Pertussis toxin treatment also restored sensitivity to catecholamines. No defect in activation by catecholamines of adenylate cyclase was seen in isolated membranes incubated in the presence of GTP. Rabbit adipocytes appear to have an excess of the inhibitory guanine nucleotide binding protein (Ni). However, in plasma membranes this protein appeared to be relatively inactive as there was an activation of adenylate cyclase activity by catecholamines in the presence of GTP. These data suggest that in intact rabbit adipocytes catecholamines and forskolin are ineffective as stimulators of adenylate cyclase due to an excess of inhibitory guanine nucleotide binding proteins.