Novel Pathway Uncovered in Diabetic Vision Loss

In people with diabetes, macular edema is a leading cause of moderate to severe vision loss. The immediate cause is increased retinal vascular permeability that allows infiltration of serum proteins and lipids into the macula. This leakage can result in thickening and interference with photoreceptor function. A study by Ben-Bo Gao, research fellow in the laboratory of Edward Feener, an HMS assistant professor of medicine at Joslin Diabetes Center, identifies a novel pathway involved in development of retinal vascular permeability and activation of the kallikrein–kinin system. The findings appear in the Jan. 28 online edition of Nature Medicine.

A path toward blindness. Retinal hemorrhage induces the kallikrein pathway, resulting in increased retinal vascular permeability and inflammation in diabetic retinopathy.

Image adapted from original courtesy of Edward Feener

Using mass spectrometry–based proteomics, Gao, Feener, and their colleagues identified 27 proteins from vitreous samples of patients with diabetic retinopathy whose levels were elevated over those of the same proteins in non-diabetic controls. The researchers homed in on carbonic anhydrase-1 (CA-1) for further study since its level was 15-fold higher than that in samples from non-diabetic controls. The scientists predicted that an overabundance of its activity in the vitreous might interfere with extracellular pH homeostasis in the neuroretina.

Intravitreal injection of human CA-1 into rats, at concentrations much lower (2 ng/µl) than those measured in vitreous samples of diabetic patients (10–50 ng/µl) led to leakage of marker fluorescein dye into the intraretinal space. This was direct evidence that CA-1, in trace quantities, increases retinal vascular permeability and induces leakage. Examination of retinal ultrastructure showed intraretinal thickening, which was similar to clinically evident edema. Screening of protease pathways led the researchers to implicate kallikrein–kinin activation in this process.

The researchers propose that retinal hemorrhage causes the release of CA-1 into the vitreous fluid from lysed red blood cells. Extracellular CA-1 mediates the hydration of CO2 released from the photoreceptor cells to bicarbonate within the vitreous. This process is normally catalyzed within the lumen of blood vessels and facilitates the removal of CO2 from the retina. The accumulated bicarbonate in the vitreous causes increasingly alkaline conditions that precede activation of the kallikrein pathway, a component of the innate inflammatory response. The outcome is the generation of bradykinin, which increases vascular permeability and inflammation.
Although retinal hemorrhage is a common finding in people with diabetic retinopathy, Feener said, the significance of this bleeding in the pathogenesis of the disorder had not been appreciated. In essence, these experiments uncover a new pathway whereby retinal hemorrhage leads to the release of CA-1 into the vitreous, which induces kallikrein–kinin activation with a consequent increase in retinal vascular permeability and edema.

There are 15 isoforms of CA in humans, and use of nonspecific CA inhibitors to treat conditions such as glaucoma often target more than one of them. The identification of extracellular CA-1 and its downstream mechanisms of action in diabetic retinopathy could enable development of targeted inhibitors for its treatment. Further studies in Feener’s laboratory are aimed at elucidating the role of other proteins occurring in the vitreous as a result of diabetic complications.

—Amita Joshi

Trans Fats May Raise Risk of Infertility

Women who have trouble conceiving often endure costly and uncomfortable invasive procedures to bring egg and sperm together. For those having problems producing a monthly egg in the first place, a condition known as ovulatory infertility, there may be an easier solution—avoid the french fries, brownies, and oversized muffins.

Jorge Chavarro, Walter Willett, and colleagues found that women who consumed two percent of their daily calories in the form of trans fat—which translates to four grams of fat for a woman eating 1,800 calories a day—exhibited a 73 percent greater chance of developing ovulatory infertility than those who consumed those calories in the form of carbohydrates. When the trans fat–consuming women were compared to women who filled that two percent slot with omega-6 polyunsaturated fats, such as canola and corn oil, the relative risk went up to 79 percent. Compared to women eating even healthier monounsaturated fats such as olive oil, trans fat–eating women had a more than twofold risk of developing ovulatory infertility. The findings appear in the January American Journal of Clinical Nutrition.

“What would happen if instead of cooking or baking with trans fats you used a healthier oil—instead of frying with margarine, you used olive oil or corn oil?” asked Chavarro, research fellow in the Department of Nutrition at HSPH. “That is more or less what we were comparing here.”

The dangers of trans fats, which are formed when vegetable oils are converted into semisolid form, are well known and recently led the New York City board of health to ban their use in restaurants. Yet no one had looked for a link with infertility. Some studies had shown that women with polycystic ovarian syndrome, who often exhibit ovulatory infertility, had more ovulations and, in some cases, got pregnant when taking medicines that activate the peroxisome proliferator–activated receptor gamma (PPAR-gamma). Though fatty acids appear to be natural ligands for the receptor, trans-fat binding has been associated with inflammation and other deleterious effects.

Spurred by these findings, Chavarro, Willett, the Frederick John Stare professor of epidemiology and nutrition at HSPH, and colleagues turned to data from the Nurses’ Health Study II. Willett and colleagues began the study in 1989, registering 116,671 women between 24 and 42 years of age. The women are followed every other year and every four years are asked to fill out an extensive questionnaire about diet. Chavarro and colleagues decided to focus on 18,555 married women, each with no previous history of infertility, who between 1991 and 1999 either became pregnant or had problems conceiving. (Married women were chosen because their pregnancies and efforts to conceive are more likely intentional.) The latter group was divided into women who attributed their problem to skipped periods (ovulatory infertility) and those who gave other reasons. The researchers identified 438 women in the ovulatory infertile group and compared them to the other two groups—those who had become pregnant or reported infertility for other reasons. Taken as a group, the 438 women with ovulatory infertility reported consuming higher levels of trans fats, though the findings must be taken with a statistical grain of salt.

“That does not mean you will be infertile or that you’re not going to be able to have children if you eat trans fats, but it means that it is going to be much harder,” Chavarro said. And the risk of developing ovulatory infertility goes up another 73 percent for every additional four grams of fat an 1,800 calorie–consuming woman eats.

“Avoid trans fats. There is really no good reason to eat them,” said Chavarro.

—Misia Landau

Negative Regulator Protein in Roundworm Controls Response to Sensory Stimuli

A protein in the regulator of G-protein signaling (RGS) family, RGS-3 is a key regulatory checkpoint in the sensory pathway that controls the response of C. elegans to external stimuli. This finding, from the lab of Anne Hart, HMS associate professor of pathology at Massachusetts General Hospital, marks the first in-depth analysis of RGS protein function in the well-characterized C. elegans sensory neurons. Conducted by lead author and postdoctoral fellow Denise Ferkey, BBS graduate student Hana Fukuto, and colleagues, the study appears in the Jan. 4 Neuron.

The researchers used a mutant C. elegans, rgs-3, to assess the role that the RGS-3 protein normally plays in G-protein signaling in response to attractive or aversive stimuli that were either strong or weak. Compared to their wild-type counterparts, mutant animals responded either slowly or poorly to strong stimuli (which included 100 percent octanol and 10 mM of quinine). Surprisingly, when the animals were exposed to diluted stimulants, the mutants responded as well as the wild-type roundworms.

Genetic and molecular analysis demonstrated that loss of RGS-3 does cause increased signaling after exposure to strong stimuli. At the molecular level, the result is an excess influx of calcium ions into sensory neurons. The researchers were able to sequester this extra calcium in the neurons by employing a calcium-binding “sponge” protein, cameleon. By thus decreasing the pool of calcium ions available for signaling, the researchers could restore responses in mutant animals. Alternatively, enhancing the synaptic transmission of glutamate from sensory neurons also rescued the behavior of rgs-3 mutant animals.

Based on these findings, the research team proposes a model whereby the excess calcium influx into the sensory neurons is detected by an unknown feedback mechanism that subsequently decreases synaptic output. The final result is a defective response observed in rgs-3 mutant animals. These observations and previous studies indicate the presence of multiple checkpoints in the sensory signaling pathway that can compensate for loss of function of individual components.

Hart, the principal investigator on the study, notes that the defective response of mutants to strong sensory stimuli was an unexpected outcome of mutating RGS-3. Since the protein is a negative regulator of G-protein signaling, a loss of its function was expected to lead to increased signaling and consequently higher neurotransmitter release, mechanisms that suggest a hyperactive response to sensory stimuli. The counterintuitive observations noted in this study, however, demonstrate that it is difficult to predict the consequences of RGS protein loss.

Further studies are under way to examine the role of additional genes that modulate behavior as a function of changes in the environment.

—Amita Joshi

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